Cotranslational protein targeting by the signal recognition particle (SRP) is a fundamental and evolutionarily conserved process that ensures accurate delivery of nascent proteins to the endoplasmic reticulum (ER). This pathway requires precise coordination between cargo recognition, targeting complex assembly, and membrane-associated handover, yet how these steps are temporally and mechanistically integrated during ongoing translation remains incompletely understood. In this thesis, I combine steady-state, pre-steady-state, and single-molecule fluorescence approaches to dissect the dynamic regulation of SRP function across distinct stages of the targeting cycle.
\r\n\r\nIn Chapter 1, I investigate how nascent polypeptide elongation modulates SRP activity. Using F\u00f6rster resonance energy transfer (FRET) measurements, I show that increasing nascent chain length enhances dynamic excursions of the signal sequence from SRP, shifting the complex into suboptimal conformations with impaired interaction kinetics with the SRP receptor (SR). Furthermore, the nascent polypeptide-associated complex (NAC) amplifies these effects, further antagonizing SRP function. These findings reveal that nascent chain length and NAC together impose a limited temporal window for efficient cotranslational targeting.
\r\n\r\nIn Chapter 2, I focus on the membrane-associated stages of the SRP pathway and elucidate the mechanism of ribosome\u2013nascent chain complex (RNC) handover to the ER translocation machinery. I demonstrate that SR remodels and partially destabilizes the SRP\u2013RNC complex by inducing signal sequence release and detachment of the SRP GTPase domain from the ribosome, while paradoxically stabilizing the overall complex through multivalent interactions. GTP hydrolysis subsequently disrupts this stabilization to enable cargo transfer. Importantly, the ER membrane selectively stabilizes a pre-handover intermediate with delayed GTP hydrolysis for correct client proteins, thereby providing a time window for continued nascent chain elongation and productive translocation.
\r\n\r\nIn Chapter 3, I explore the expression of the Sec61 complex in insect cells and its reconstitution into nanodiscs to recapitulate the effects of the ER membrane on RNC handover and SRP conformation. Using a copolymer-based method, I successfully purified Sec61-containing nanodiscs comprising all three subunits. However, further assays are required to verify the structural and functional integrity of the reconstituted Sec61 translocon, and additional optimization of the purification strategy is needed.
\r\n\r\nTogether, this work advances our understanding of the molecular mechanisms underlying SRP-dependent cotranslational targeting. In conjunction with prior studies from our laboratory, it contributes to a high-resolution mechanistic model of this pathway. More broadly, it reveals how multiple regulatory layers are temporally coordinated: how SRP selectively rejects RNCs bearing excessively elongated nascent chains to enhance targeting specificity, and how GTPase activity serves as a molecular timer to ensure cargo handover occurs at the appropriate stage of nascent chain elongation.
", "doi": "10.7907/tr6n-t650", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18677", "collection": "thesis", "collection_id": "18677", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282026-211327832", "type": "thesis", "title": "Optimizing Treslin-MTBP for Cryo-EM Analysis", "author": [ { "family_name": "Ng", "given_name": "Cai Tong", "orcid": "0000-0002-1082-8667", "clpid": "Ng-Cai-Tong" } ], "thesis_advisor": [ { "family_name": "Dunphy", "given_name": "William G.", "orcid": "0000-0001-7598-8939", "clpid": "Dunphy-W-G" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Glover", "given_name": "David M.", "orcid": "0000-0003-0956-0103", "clpid": "Glover-D-M" }, { "family_name": "Dunphy", "given_name": "William G.", "orcid": "0000-0001-7598-8939", "clpid": "Dunphy-W-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA replication initiation is a highly regulated process in eukaryotes to maintain genome integrity. Misfiring of the DNA replication machinery from replication origins can result in gene amplification and genome instability. In budding yeast, ORC, Cdc6, Cdt1, and Mcm2-7 assemble on the sites of origins to form the inactive pre-replicative complex. Next, phosphorylation of Sld2 and Sld3 causes them to interact with Dbp11, leading to the recruitment of Cdc45 and GINS to the pre-replicative complex, which results in its activation. The vertebrate homolog of Sld3 is Treslin. Treslin is tightly associated with MTBP throughout the cell cycle, and the complex is essential for DNA replication in metazoan systems, including Xenopus egg extracts and human cells. Here, I aim to understand the mechanism of Treslin-MTBP function by studying its molecular structure. I established purification protocols to produce the Treslin-MTBP complex at high concentration. I then studied the overall structure of the complex using cryo-electron microscopy and obtained reconstructions of the complex at ~20 \u00c5. The reconstructions show that the Treslin-MTBP complex exhibits a large variety of conformations, indicating its intrinsic flexibility. The complex is composed of large, stable domains that can be observed in 2D classes. Using cross-link mass spectrometry, I also showed that Treslin-MTBP transiently binds to Mcm4 in cells, making Mcm4 a potential binding partner for future structural studies of the complex.", "doi": "10.7907/p11y-7r94", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:18754", "collection": "thesis", "collection_id": "18754", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012026-215117772", "type": "thesis", "title": "Characterizing the Lipid II Flippase MurJ as an Antibiotic Target", "author": [ { "family_name": "Li", "given_name": "Yancheng", "orcid": "0000-0002-7110-2448", "clpid": "Li-Yancheng" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The rapid emergence of antibiotic resistance, coupled with a decline in antibiotic discovery, poses a major threat to global public health and underscores the urgent need for the identification and characterization of new antibiotic targets and mechanisms. The lipid II flippase MurJ, an essential membrane transporter that flips the peptidoglycan precursor lipid II across the cytoplasmic membrane, represents a validated yet unexploited antibiotic target. MurJ has been repeatedly targeted by both bacteriophage-encoded lysis proteins and by small-molecule screening efforts. Distinct single gene lysis protein (Sgls) from small lytic phages have evolved independently to inhibit MurJ, suggesting that MurJ represents a key step for killing bacteria and an evolutionarily validated target. Structural and mechanistic understanding of MurJ has largely been derived from diderm bacteria. However, small molecule compounds that target MurJ have been identified specifically in monoderm pathogens, and the corresponding monoderm MurJ homologs remain uncharacterized. Despite strong validation of MurJ as a potential antibiotic target, the inhibition mechanisms are unknown, limiting efforts to translate this key step into effective therapeutic strategies. In this thesis, we define the inhibition mechanisms of MurJ by Sgls using structural approaches. We show that distinct Sgls converge on a common interface on MurJ and trap MurJ in a periplasm-open conformation. We further identified and structurally characterized a novel MurJ-targeting Sgl from the predicted phage Changjiang3, supporting convergent evolution toward a shared inhibition mechanism. In addition, we determined structures of monoderm MurJ in multiple conformational states, revealing both conserved and divergent features for the transport mechanism across monoderms and diderms. Together, these findings highlight new opportunities for targeting MurJ for the development of novel antimicrobials", "doi": "10.7907/bqpa-4195", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17738", "collection": "thesis", "collection_id": "17738", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10302025-130729385", "primary_object_url": { "basename": "Mahajan_thesis_final_submission.pdf", "content": "final", "filesize": 97288552, "license": "other", "mime_type": "application/pdf", "url": "/17738/1/Mahajan_thesis_final_submission.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanistic Studies of ArsA ATPase and ArsB Transporter of the Bacterial Arsenite Efflux System", "author": [ { "family_name": "Mahajan", "given_name": "Shivansh", "orcid": "0000-0002-3041-7988", "clpid": "Mahajan-Shivansh" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Arsenic is a notorious metalloid that contaminates the groundwater in several regions worldwide. The trivalent state of arsenic \u2013 arsenite (AsIII) \u2013 is the abundant species of arsenic under reducing conditions of subsurface waters, and is readily mobilized in aqueous environments. This exposes organisms to toxic concentrations of the metalloid. AsIII is particularly toxic to living systems due to its ability to form stable polar covalent bonds with exposed thiol groups, thus disrupting protein structure and function. Arsenic detoxification systems such as efflux pumps, exist in most organisms that confer tolerance to toxic concentrations of arsenicals found in their environment. The ars operon in many bacteria and some archaea confers resistance to AsIII via ArsB, an integral membrane transporter and ArsA, a cytoplasmic P-loop ATPase. These proteins, collectively referred to as the 'ArsAB efflux pump', facilitate toxic AsIII export in an ATP-dependent manner. In addition, ArsB can operate by itself as a proton-coupled secondary transport and confer intermediate levels of AsIII resistance. The mechanisms of this dual mode of AsIII efflux are poorly understood, particularly the molecular events associated with the capture of AsIII from the cytoplasm by ArsA, its transfer to ArsB and subsequent vectorial transport across the membrane. Apart from understanding fundamental mechanisms of toxic metalloid detoxification in living systems, molecular-level investigations of AsIII efflux systems are of broad biotechnological interest for their potential to inform robust and sustainable bioremediation strategies. In this thesis, we elucidate the mechanism of ArsA ATPase and ArsB transporter using structural approaches. We characterized the nucleotide hydrolysis mechanism of ArsA by single particle cryogenic electron microscopy (cryo-EM), outlining various conformational states of the ATPase that modulate the nucleotide-dependent capture and delivery of AsIII for efflux. We show that this mechanism is consistent with the general mechanistic framework of the Intradimeric Walker A (IWA) family of ATPases. Furthermore, overexpression and purification of the membrane transporter ArsB enabled characterization of the first structure of ArsB by cryo-EM, in both apo and AsIII-bound states. Lastly, we show that ArsB enhances steady-state ATPase activity of ArsA, indicating a direct interaction between the two components of the efflux pump. Computational modeling gives some insights into a putative ArsAB interaction interface. While several mechanistic questions remain, the findings reported in this thesis together constitute a foundation for future mechanistic elucidation of the ArsAB efflux system.", "doi": "10.7907/xksn-7t38", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:16592", "collection": "thesis", "collection_id": "16592", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07252024-205053475", "primary_object_url": { "basename": "Thesis_Doris_Mai.pdf", "content": "final", "filesize": 46775444, "license": "other", "mime_type": "application/pdf", "url": "/16592/6/Thesis_Doris_Mai.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Advancing Structural Analysis with Computational Methods Development", "author": [ { "family_name": "Mai", "given_name": "Huanghao", "orcid": "0000-0003-2278-0768", "clpid": "Mai-Huanghao" } ], "thesis_advisor": [ { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In this thesis, a set of computational methods is developed to extend structural techniques beyond their conventional practice. First, we build in silico simulations and image processing protocols to design a new data acquisition workflow in cryo-electron tomography. This enables in situ visualization of macromolecular complexes at sub-nanometer resolution in a micron-scale field of view. Then, we demonstrate the applicability of a novel machine-learning algorithm in processing small molecule electron diffraction data for the first time. For most molecules tested, the correct ab initio structures can be obtained without the common practice of manual dataset curation. Finally, molecular dynamics simulations using crystallographic structures of protein and drug molecule complexes are performed to investigate the fundamental principles of a ternary binding property. A minimal forcefield with multi-scale coarse-graining enables alchemical free energy calculations at an unconventional size of perturbation while providing physical insight into the role of the drug linker and protein shapes.", "doi": "10.7907/4p95-d385", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17027", "collection": "thesis", "collection_id": "17027", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02252025-210356380", "type": "thesis", "title": "Direct Visualization of Cellular Protein Complexes in situ by Fluorescence-Guided Cryo-FIB-SEM and Cryo-ET", "author": [ { "family_name": "Wang", "given_name": "Jue (Phyllis)", "orcid": "0000-0002-5623-0994", "clpid": "Wang-Jue-Phyllis" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cryogenic electron tomography (cryo-ET) is a technique that can reconstruct three-dimensional volumes of large protein complexes in situ at sub-nanometer resolution. In addition to imaging proteins extracted from cells, cryo-ET also allows direct visualization of macromolecular complexes in their native environment. To reveal molecular details buried deeply inside thick eukaryotic cells, cryogenic focused ion beam milling with scanning electron microscopy (cryo-FIB-SEM) has been established as the leading approach for preparing thin sections of cells suitable for cryo-ET. Recent advances in cryo-FIB-SEM systems integrate fluorescence microscopy (cryo-FM-FIB-SEM) to help direct the milling to specific labeled regions of interest. This method has had success localizing large organelles and protein aggregates. Unfortunately, it is difficult to localize small and rare targets along the optical axis of the cryo-FIB. This thesis work pioneered a customized integrated tri-coincident imaging system (ENZEL) that allows for simultaneous fluorescence imaging and cryo-FIB milling. This novel method allows precise targeting of small and rare structures with a high success rate compared to other systems. To demonstrate the imaging workflow, we applied this approach to visualize the microtubule organizing center (MTOC), a crucial organelle responsible for cell division and cellular transport in mammalian cells. It presents as a single fluorescent punctum expanding approximately 1 \u00b5m in diameter in live cells, making it a challenging target for cryo-FM-FIB-SEM. Our cryo-tomograms resolved the molecular architecture of the MTOC and revealed molecular details at the microtubule nucleation sites. We then used the ENZEL to explore more complicated biological systems. Here we chose the NLRP3 inflammasome, a master mediator of innate immunity colocalized with the MTOC. We captured the first in-situ image of the NLRP3 inflammasome and showed new mechanistic insights that this complex forms a condensate at the MTOC, halting cell division and inducing drastic organelle changes.", "doi": "10.7907/rmdd-5t54", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17303", "collection": "thesis", "collection_id": "17303", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302025-004839918", "primary_object_url": { "basename": "Thesis_Maryann_Morales.pdf", "content": "final", "filesize": 5588826, "license": "other", "mime_type": "application/pdf", "url": "/17303/2/Thesis_Maryann_Morales.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Metal Binding to Nsp1, a SARS-CoV-2 Protein", "author": [ { "family_name": "Morales", "given_name": "Maryann", "orcid": "0000-0002-1778-8901", "clpid": "Morales-Maryann" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Hadt", "given_name": "Ryan G.", "orcid": "0000-0001-6026-1358", "clpid": "Hadt-Ryan-G" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "orcid": "0000-0002-4453-9716", "clpid": "Winkler-J-R" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Virgil", "given_name": "Scott C.", "orcid": "0000-0001-8586-5641", "clpid": "Virgil-S-C" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The COVID-19 pandemic, caused by SARS-CoV-2, has underscored the need for novel antiviral strategies beyond vaccines. A key virulence factor in SARS-CoV-2 is nonstructural protein 1 (Nsp1), which suppresses host immune responses by degrading mRNA, inhibiting nuclear export, and binding to the 40S ribosomal subunit to block host translation. Its intrinsically disordered C-terminal domain complicates structure-based drug design, prompting exploration of alternative approaches.
\r\n\r\nThis work investigates the use of transition metal coordination to target disordered regions of Nsp1. Copper(II) and cobalt(III) complexes were examined for their ability to bind histidine residues\u2014particularly H165, critical for ribosome interaction. Biophysical techniques, including fluorescence spectroscopy, EPR, and \u2075\u2079Co NMR, along with computational modeling, were used to characterize binding to Nsp1-derived peptides and the full-length protein.
\r\n\r\nCu(II) displayed pH-dependent coordination through histidine and backbone amides, while oxidized Co(III) complexes formed stable, substitution-inert interactions. Multi-site binding and distinct kinetic profiles were observed. In vitro translation assays showed that metal complexes can affect translation, though selective inhibition of Nsp1 remains challenging.Overall, this work provides a foundation for targeting disordered viral proteins using coordination chemistry.
", "doi": "10.7907/z9m9-yn02", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17288", "collection": "thesis", "collection_id": "17288", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282025-222913964", "primary_object_url": { "basename": "VyNguyen_Thesis_Final_proofread.pdf", "content": "final", "filesize": 19155203, "license": "other", "mime_type": "application/pdf", "url": "/17288/1/VyNguyen_Thesis_Final_proofread.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structural and Mechanistic Studies of Membrane Protein Biogenesis and Quality Control at the Endoplasmic Reticulum", "author": [ { "family_name": "Nguyen", "given_name": "Vy Ngoc Mai", "orcid": "0000-0002-7563-7386", "clpid": "Nguyen-Vy-Ngoc-Mai" } ], "thesis_advisor": [ { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Membrane proteins make up around 30% of the human proteome and carry out essential functions in the cell, including but not limited to signaling, cell adhesion, and metabolic transport. To maintain homeostasis, the eukaryotic cell has evolved complex pathways for membrane protein biogenesis and quality control, as they contain hydrophobic transmembrane domains (TMDs) that can easily aggregate in the aqueous environment of the cytosol causing cellular damage. At the endoplasmic reticulum (ER), the ER membrane protein complex (EMC) co-translationally inserts the first TMD of multipass membrane protein and post-translationally inserts tail-anchored membrane proteins. In this thesis, we are able to show how the EMC can coordinate with the other protein machineries of the multipass translocon (such as the back-of-Sec61/BOS complex) at the ER to accommodate the insertion of diverse multipass membrane protein substrates, depending on the biophysical properties of their N-terminal soluble domain. We also structurally characterize the EMC\u2022BOS holocomplex, highlighting its spatial relation with the other biogenesis factors at the multipass translocon. In addition, this thesis also explores how a novel quality control factor TXNDC15 functions in ER-associated degradation (ERAD) to facilitate degradation of unassembled membrane proteins. We found that TXNDC15 works with the E3 ligase MARCHF6 and recognizes a set of membrane proteins with exposed hydrophobic domain in the ER lumen. Lastly, we were able to also link TXNDC15\u2019s quality control function to its implication in ciliopathies, such as Meckel-Gruber syndrome.", "doi": "10.7907/ar35-w658", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16607", "collection": "thesis", "collection_id": "16607", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08022024-005547280", "type": "thesis", "title": "Studies on Scaling Throughput in Protein Engineering", "author": [ { "family_name": "Schaus", "given_name": "Lucas Jean Nicolas", "orcid": "0000-0002-6094-7402", "clpid": "Schaus-Lucas-Jean-Nicolas" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "Resnick Sustainability Institute" }, { "literal": "div_chem" } ], "abstract": "In this work we present three studies in protein engineering. While all three protein classes that have been targeted for engineering tasks are very different, the studies have a focus on scaling-up the throughput in protein engineering.
\r\n\r\nThe first study concerns machine learning (ML) based antibody humanization techniques. Achieving a reduction of patient anti-drug antibody responses in clinical trials is the goal of antibody humanization. To measure this however, one needs to pass significant scientific, bureaucratic, and financial hurdles, which is very rarely done and especially never at scale. Most existing ML-based antibody humanization techniques claim that they work without providing any experimental evidence. We developed Mousify as an in silico antibody humanization platform to place existing models into one framework for wet-laboratory validation. We demonstrate that even the best models have a fundamental flaw in that they only generate a single antibody. We use Mousify and Markov chains to show that using ML-based antibody humanization models for library generation is not only feasible but produces both stable and functional variants. Learning the lessons from our wet-laboratory experiments, we then developed a variational autoencoder model with properties that hopefully improve the outcomes of antibody humanization experiments.
\r\n \r\nIn the second study, we outline our plans and initial results to develop a bioelectrocatalytic system for the conversion of N2 to ammonia using nitrogenase. Most of the world\u2019s ammonia is used for agricultural purposes and is produced via the environmentally damaging Haber-Bosch process. Engineering nitrogenase for the bioelectrocatalytic production of ammonia is not trivial and a high throughput is not guaranteed. We present preliminary results in how throughput can be increased through diazotrophic pre-selection of nitrogenase variants, as well as a quest to find the ideal starting point for engineering using a combination of ancestral sequence reconstruction and generative protein language models.
\r\n\r\nIn the third and final study we present a directed evolution campaign to evolve protoglobins for the enantioselective catalytic formation of cis-trifluoromethyl substituted cyclopropanes, the first such reaction in both the chemical and biological world. Not only is the enzyme ApePgb LQ capable of efficiently performing carbene insertions into double-bonds, but it also shows a much more diverse substrate scope than similar enantioselective formations of trans-trifluoromethyl substituted cyclopropanes. After demonstrating that ApePgb LQ reactions can be increased to a 1-mmol scale, we investigated the nature of protoglobin cis-selectivity using various computational methods.
", "doi": "10.7907/jqng-x012", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16249", "collection": "thesis", "collection_id": "16249", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11172023-202811116", "type": "thesis", "title": "Development of Microcrystal Electron Diffraction Techniques for the Characterization of Small Molecules and Novel Materials", "author": [ { "family_name": "Jones", "given_name": "Christopher Glenn", "orcid": "0000-0003-4308-1368", "clpid": "Jones-Christopher-Glenn" } ], "thesis_advisor": [ { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Manthiram", "given_name": "Karthish", "orcid": "0000-0001-9260-3391", "clpid": "Manthiram-Karthish" }, { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Traditional techniques for structural analysis, such as X-ray crystallography and Nuclear Magnetic Resonance (NMR), have been invaluable in understanding the composition of various substances. However, these methods often encounter challenges when applied to the analysis of small molecules and certain novel materials, particularly those that cannot form large, high-quality crystals. The research presented here focuses on the evolution and applications of Microcrystal Electron Diffraction (MicroED), a transformative technique that has expanded the boundaries of structural analysis. We trace the developmental trajectory of MicroED, exploring its underlying principles, technological advancements, and comparative advantages over conventional methods. A variety of data from several key studies was collected through a series of experiments utilizing MicroED to analyze a range of substances, from small organic molecules to complex novel materials and innovative inorganic complexes. MicroED offers unprecedented resolution and sensitivity, capable of structural elucidation where other methods fail. In particular, MicroED has been successful in determining the structures of several novel materials and small molecules with applications in areas such as renewable energy, advanced manufacturing, and pharmaceuticals. Furthermore, this technique is highly amenable to integration with other analytical and computational methods, including machine learning algorithms for data interpretation, enhancing its applicability and efficiency. This research contends that MicroED is not merely an alternative but a substantial upgrade to existing methodologies, holding the potential to revolutionize fields as diverse as materials science, chemistry, and medicine.", "doi": "10.7907/cfnr-f362", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16241", "collection": "thesis", "collection_id": "16241", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11062023-233057820", "type": "thesis", "title": "Using Functional Genomics to Characterize Biogenesis and Quality Control Pathways in the Mammalian ER", "author": [ { "family_name": "Page", "given_name": "Katharine-Rose", "orcid": "0000-0001-8904-1244", "clpid": "Page-Katharine-Rose" } ], "thesis_advisor": [ { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "thesis_committee": [ { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cells are tasked with ensuring the proper synthesis, localization, and insertion of membrane proteins at the mammalian endoplasmic reticulum (ER). Recent advances have shown that the insertion of transmembrane domains into the ER and the translocation of their associated soluble domains across the ER can be facilitated by members of the Oxa1 superfamily of insertases. The ER membrane protein complex (EMC) contains an Oxa1 insertase that facilitates the co-translational insertion of the first TMD of Nexo proteins, which have their N-terminal soluble domains localized in the ER lumen or extracellular space. Additionally, recent work has described the multipass translocon, a supercomplex at the Sec61 translocation channel that facilitates insertion of multipass membrane proteins. During my PhD and in collaboration with other scientists in the Voorhees lab, I elucidated how the EMC cooperates with the multipass translocon to facilitate biogenesis of multipass membrane proteins. We took a systematic approach, applying a combination of functional genomics, biochemistry, structural biology, and mechanistic cell biology to understand how the biophysical properties of the TMDs and the intervening soluble domains of multipass membrane proteins influence their path into the ER bilayer. We show that the EMC is epistatic with members of the multipass translocon, including the BOS, GEL, and PAT complexes. We structurally characterize the EMC\u2022BOS holocomplex, showing that these complexes directly interact, and that this interaction is mutually exclusive to the interaction of BOS and Sec61. Further, we demonstrate that Nexo proteins that contain a net positive charge in their N-terminal soluble domain are difficult for the EMC to insert and thus also rely on Sec61 or TMCO1, the Oxa1 insertase of the GEL complex, for insertion. We overturn the prevailing model for multipass membrane protein insertion and show that how this diverse class of membrane proteins utilizes the suite of ER biogenesis machinery depends on their distinct biophysical properties. In addition to biogenesis, during my PhD I also studied how the cell surveils multi-subunit complex assembly. I focused on a model ER-resident and obligate complex. These subunits are unstable and degraded in the absence of their binding partner, but how they are recognized for degradation is unknown. I used unbiased functional genomics approaches to identify the quality control components that regulate orphan subunit degradation in the cell. Further, I used proteomics to identify endogenous substrates of this particular ERAD pathway. This work expands upon our understanding of how multi-subunit complexes are regulated by machinery in the cell.", "doi": "10.7907/4c7k-yw62", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16213", "collection": "thesis", "collection_id": "16213", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10232023-184021847", "primary_object_url": { "basename": "saladi-dissertation.pdf", "content": "final", "filesize": 327264663, "license": "other", "mime_type": "application/pdf", "url": "/16213/1/saladi-dissertation.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Some Computer Studies of Membrane Proteins, Molecular Chaperones, and Color", "author": [ { "family_name": "Saladi", "given_name": "Shyam Madhukar", "orcid": "0000-0001-9701-3059", "clpid": "Saladi-Shyam-Madhukar" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis shares a series of stories on seemingly disparate topics united by my efforts and love of computers. Initially, I discuss how the challenge of membrane protein expression provided an initial impetus for research. I channeled efforts towards developing a predictive (machine-learning) model for heterologous overexpression in E. coli. While we made strides to extend this model to other systems (not discussed here), my time was refocused onto questions of more fundamental biochemical interest: the biogenesis of tail-anchored membrane proteins. I built structural, predictive, and phylogenetic models to better understand how the C-terminal domain of co-chaperone Sgt2 functioned, refined the definition of the wider Sti1 family which includes Sgt2-C, and extended our understanding of those features of tail-anchored proteins that determine successful targeting in Yeast and Human cells. I developed a deep phylogeny of Get3, a chaperone involved in tail-anchored protein biogenesis, and helped specifically place Get3 proteins of photosynthesising organisms into evolutionary context. Along the way, I developed a parallel and compelling theme around data visualization, specifically around the use of colormaps across the life sciences. In particular, I built an application to screen and notify preprint authors when their manuscript had poor colormap usage. This was the first time automated software has been used to help authors improve their work at the preprint stage, an area that has grown significantly since my initial work. Finally, I brought together structural biology and data visualization by making perceptually uniform colormaps available in popular molecular visualization software tools to advocate for more thoughtful color usage in the field.", "doi": "10.7907/40cw-kn70", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16474", "collection": "thesis", "collection_id": "16474", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-022957246", "type": "thesis", "title": "Partial Synthetic Models of the FeMoco Nitrogenase Cluster with Bridging C-Based Ligands", "author": [ { "family_name": "Le", "given_name": "Linh Nguyen Vuong", "orcid": "0000-0003-1451-2675", "clpid": "Le-Linh-Nguyen-Vuong" } ], "thesis_advisor": [ { "family_name": "Agapie", "given_name": "Theodor", "orcid": "0000-0002-9692-7614", "clpid": "Agapie-T" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "Garnet K.", "orcid": "0000-0001-8009-6038", "clpid": "Chan-G-K" }, { "family_name": "Agapie", "given_name": "Theodor", "orcid": "0000-0002-9692-7614", "clpid": "Agapie-T" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biological N\u2082 reduction to NH\u2083 occurs in microorganisms using the enzyme nitrogenase. This complex system consists of several iron-sulfur clusters, where the active site contains a MFe\u2087S\u2089C cluster (M = Mo, V, Fe) known as FeM cofactor (FeMco). The cluster includes an unusual interstitial carbide ligand, which is rare in both inorganic chemistry and biology. In addition, the role of this motif within the enzyme is not well-understood, and studies on synthetic model complexes are limited due to the absence of any previously reported iron-sulfur cluster systems bearing a carbon-based ligand that bridges the Fe atoms. Thus, this thesis focuses on developing strategies to insert a bridging carbon-based ligand into an iron-sulfur cluster platform.
\r\n \r\nChapter 1 provides a general introduction and overview of complex biologically relevant iron-sulfur clusters and their corresponding synthetic analogs, with focus on NiFe CO dehydrogenase (CODH), acetyl CoA synthase (ACS), [FeFe] hydrogenase, P-cluster, and M-cluster of nitrogenase.
\r\n\r\nChapter 2 discusses the formation of a cluster with a \u03bc\u2083-carbyne ligand resulting from the ring-opening of a bisaminocyclipropenylidene ligand. Electrochemical studies on this system and related species suggest that a chelating \u03bc\u2083-carbyne leads to clusters with highly negative reduction potentials compared to \u03bc\u2083-N or S ligands, suggesting that the interstitial carbide in FeMco may play a role in modulating the redox potential of the cluster to allow for the reduction of difficult substrates like N\u2082.
\r\n\r\nChapter 3 focuses on the binding of CO to the cluster with a \u03bc\u2083-carbyne fragment, resulting in a high level of CO activation at 1851 cm\u207b\u00b9 in the neutral cluster and 1782 cm\u207b\u00b9 in the reduced cluster, Computational studies suggest that the bridging carbyne stabilizes the intermediate spin state at the Fe sites, resulting in more electrons in orbitals that can backbond with CO and greater activation. This suggests that the carbide in FeMco might play a role in modulating the electronic structure at the Fe sites to allow for greater activation of substrates.
\r\n\r\nChapter 4 highlights the synthesis of a cluster bearing a \u03bc\u2084-carbide ligand using a previously reported terminal Mo carbide complex, with a bridging CO ligand that resembles the lo-CO form. The S = 1/2 spin state provides an opportunity to study the metal-carbon interaction by pulse EPR spectroscopy.
\r\n\r\nIn Chapter 5, a cluster ligated by an anthracene-bridged bisphenoxide ligand is described. Upon reduction, the anthracene bridge moves closer to one Fe site and interacts with it in an \u03b7\u00b2 manner. This species can catalyze the electrochemical reduction of proton to form H2, possibly through a protonated cluster intermediate. The studies demonstrate the ability of the cluster to catalyze a biologically relevant reaction, and possibility for future studies on protonated species that have only been proposed in reactions of synthetic iron-sulfur clusters.
", "doi": "10.7907/9xgc-d212", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16280", "collection": "thesis", "collection_id": "16280", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01162024-161326655", "primary_object_url": { "basename": "TDilanyan_Thesis.pdf", "content": "final", "filesize": 7266593, "license": "other", "mime_type": "application/pdf", "url": "/16280/4/TDilanyan_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Open-Source Custom Beads for Single-Cell Transcriptomics", "author": [ { "family_name": "Dilanyan", "given_name": "Taleen Gaied", "orcid": "0000-0002-3131-3259", "clpid": "Dilanyan-Taleen-Gaied" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Open-source single-cell genomics technologies have helped democratize single-cell genomics and expedite method development. Methods such as inDrops and Drop-seq for single-cell RNA-seq preceded popular technologies such as the 10x Genomics\u2019 Chromium platform, however despite initial enthusiasm for open-source methods, their popularity has waned. A major reason has been the lack of availability of low-cost, customizable beads, which are essential for microfluidics based single-cell RNA-seq. We address this challenge by introducing a new method for producing barcoded hydrogel beads for single-cell RNA-seq called HiPER (High-throughput PER-barcoded hydrogel beads) that allows for increasing the diversity of barcode sequences, reducing manufacturing cost, and that can be readily adapted to custom applications. HiPER barcodes are decoupled from the capture sequences and can therefore be configured to capture RNA, DNA, or tailored for specific-gene enrichment.", "doi": "10.7907/v52p-gf80", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16473", "collection": "thesis", "collection_id": "16473", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032024-011201674", "primary_object_url": { "basename": "Mengtong Duan 2024 Jun 14.pdf", "content": "final", "filesize": 35875270, "license": "other", "mime_type": "application/pdf", "url": "/16473/1/Mengtong Duan 2024 Jun 14.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Expanding Frontiers in Biomedical Imaging and Synthetic Biology: Dynamic Acoustic Reporter Gene Imaging and Ratio-Tuning of Mammalian mRNA Polycistronic Expression", "author": [ { "family_name": "Duan", "given_name": "Mengtong (Tom)", "orcid": "0000-0002-1601-8876", "clpid": "Duan-Mengtong-Tom" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis presents a comprehensive exploration of the next generation of mammalian Acoustic Reporter Genes (mARGs), unveiling a novel approach for non-invasive, real-time imaging of cellular processes and gene expression within live animals1. Building on the foundational work of first-generation ARGs2,3, which introduced the groundbreaking concept of using gas vesicle (GV) genes as genetically encoded ultrasound contrast agents, this research tackles the inherent limitations of these pioneering systems. The first segment details the development and characterization of the second-generation mARGs which significantly improve upon their predecessors by offering robust expression without the need for monoclonal screening, dynamic non-destructive imaging capabilities, and customizable acoustic properties through gene and protein level modifications. This advancement not only enhances the utility of mARGs in biomedical imaging but also paves the way for their application in novel therapeutic monitoring strategies, as exemplified by real-time tracking of tumor development and ultrasound-guided tumor biopsies that leverage gene expression information.
\r\n\r\nFurther, the thesis delves into the structural, genetic, and biochemical principles underpinning GV assembly, addressing a critical knowledge gap that has persisted despite the utility of GVs in ultrasound imaging. Understanding these assembly mechanisms is crucial for the engineering of improved ARGs.
\r\n\r\nThe exploration then extends into innovative bioengineering methodologies, specifically Stoichiometric Expression of Messenger Polycistrons by Eukaryotic Ribosomes (SEMPER), a synthetic biology breakthrough enabling the expression of multiple proteins at precise stoichiometries from single, compact transcripts.4 SEMPER represents a strategic advancement in the field, facilitating efficient formation of multi-protein complexes, minimizing cellular toxicity, and broadening the scope of potential applications in genetic engineering, including the creation of enhanced cell lines and circuits for research and therapeutic purposes.
\r\n\r\nCollectively, this work not only advances our understanding of GV-based ultrasound imaging and gene expression tracking but also introduces versatile genetic tools for the manipulation of cellular machinery. These achievements mark significant strides in the fields of synthetic biology and molecular imaging, setting the stage for future innovations in non-invasive diagnostics, cellular therapy, and cancer monitoring research. Through the integration of improved acoustic reporter genes, insights into gas vesicle assembly, and the SEMPER method for gene expression, this thesis embodies a holistic approach to overcoming current challenges and unlocking new potentials in biomedical engineering and synthetic biology.
", "doi": "10.7907/avgk-yc71", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16279", "collection": "thesis", "collection_id": "16279", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01122024-002204703", "primary_object_url": { "basename": "Denman_W_thesis_final.pdf", "content": "final", "filesize": 9675541, "license": "other", "mime_type": "application/pdf", "url": "/16279/1/Denman_W_thesis_final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Birth of PICL: New Laboratory Experiments for Understanding Ocean Worlds", "author": [ { "family_name": "Denman", "given_name": "William Thomas Plent", "orcid": "0000-0003-4752-0073", "clpid": "Denman-William-Thomas-Plent" } ], "thesis_advisor": [ { "family_name": "Brown", "given_name": "Michael E.", "orcid": "0000-0002-8255-0545", "clpid": "Brown-M-E" } ], "thesis_committee": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Brown", "given_name": "Michael E.", "orcid": "0000-0002-8255-0545", "clpid": "Brown-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A laboratory set up was built analogous to that of the environment of the Galilean system. Previous work has focused on vapor deposition and shied away from bulk samples which more closely resemble the surface of Europa. The focus of this research has been the relationship between laboratory data to observational spectra collected. Data from various telescopes has given an indication of the species which exist on the surface of Europa. Linear spectral modeling has not been effective in identifying these species due to a lack of viable candidates.\r\nChapter II focuses on the instrumentation and sample preparation for the laboratory set up. In Chapter III data is presented on the irradiation of sodium chloride at Europa like conditions and the features that arise with cryogenic irradiation. This data is compared to observational data from HST and provides strong evidence for the presences of sodium chloride (NaCl) on Europa's leading hemisphere. Chapter IV presents FTIR and UV/VIS data for the irradiation of sulfate salts suspected to be present on Europa. This presents one of the first instances of cyrogenic electron irradiation of sulfates compared to new data from JWST. Mechanisms for the trapping of carbon dioxide at both Europa and Ganymede have also been investigated. These experiments are paramount for understanding the composition of Europa\u2019s ocean and can be utilized by the Europa Clipper team. Future experiments involving laboratory spectroscopy of carbon dioxide trapping are also highlighted. The use of cyrogenic gamma irradiation experiments and their feasibility are explored.", "doi": "10.7907/erq7-vb46", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16259", "collection": "thesis", "collection_id": "16259", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092023-142606831", "type": "thesis", "title": "Characterization of a Novel Membrane Protein Insertase in the Mitochondrial Outer Membrane", "author": [ { "family_name": "Stevens", "given_name": "Taylor Anthony", "orcid": "0000-0002-6232-5316", "clpid": "Stevens-Taylor-Anthony" } ], "thesis_advisor": [ { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Mitochondria are eukaryotic organelles derived from the endosymbiosis of an ancient bacteria. As a result of their endosymbiotic origin, the mitochondrial proteome is composed of a mixture of ancient bacterial derived genes and others which are unique to eukaryotes. This dual bacterial/eukaryotic protein origin results in a complicated landscape for biogenesis of mitochondrial proteins. This is particularly true for mitochondrial membrane proteins, since mitochondria have both an inner and outer membrane each with unique protein composition. Proteins localized to the outer mitochondrial membrane (OMM) are of particular interest due to their connection to many important physiological pathways in humans. OMM proteins are known to be inserted into the lipid bilayer by the MIM complex in yeast and by ATOM36 in trypanosomes, however it is not known how they are inserted in human cells. In my Ph.D. thesis, I describe the development of improved biochemical tools for protein purification and characterization, and then use them as part of an effort to characterize MTCH2, which we identify as the human gene responsible for OMM protein insertion. After identifying MTCH2 in a genome-wide screen, we use a variety of cell biology and biochemical experiments to show that MTCH2 is both necessary and sufficient for OMM protein insertion. We further show that endogenous OMM proteins are affected by MTCH2 depletion, and that apoptosis, a pathway relying on OMM proteins, is sensitive to MTCH2 modulation. Additional work in my thesis demonstrates that MTCH2 is a deeply conserved gene across metazoans, that other OMM insertases likely evolved independently in separate multi-cellular eukaryotic lineages.", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16088", "collection": "thesis", "collection_id": "16088", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062023-051752771", "primary_object_url": { "basename": "maggiolo_ailiena_2023.pdf", "content": "final", "filesize": 143058996, "license": "other", "mime_type": "application/pdf", "url": "/16088/1/maggiolo_ailiena_2023.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Crystallographic Studies of Nitrogenase", "author": [ { "family_name": "Maggiolo", "given_name": "Ailiena Okumura", "orcid": "0000-0003-1707-5060", "clpid": "Maggiolo-Ailiena-Okumura" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nitrogenase is the only enzyme known to reduce atmospheric dinitrogen to ammonia, producing a biologically available form of nitrogen. The primary component of nitrogenase, the molybdenum-iron (MoFe) protein, binds and turns over substrate after reduction by multiple electron equivalents, which are supplied by the obligate reductase, the iron (Fe) protein. Previous high-resolution X-ray crystal structures have provided pictures of the nitrogenase proteins and revealed the geometry of their metalloclusters. In this thesis, we study MoFe protein crystal isoforms and their crystal pathologies to classify isomorphous candidates suitable for crystallographic merging. We then leverage this classification to determine a high-resolution structure of the MoFe protein with improved geometric accuracy and lower coordinate error than currently available models. The reduced states of the MoFe protein are challenging to capture and therefore have been minimally characterized. We explore the structural consequences of introducing reductants and oxidants into MoFe protein crystals and study the effects of X-ray induced photoreduction on the metalloclusters. Further, we determine the radiation damage-free X-ray crystal structure of MoFe protein. The Fe protein adopts various conformational states as it functions in ATP-coupled electron transfer to the MoFe protein. We examine a set of proteins that are evolutionarily related to the Fe protein and have diverse functionalities, but retain similarity in their ATP-dependent function and allostery as a result of their conserved structural features. Finally, we characterize the structural and functional aspects of the Fe protein lacking an iron-sulfur cluster. These studies expand our understanding of the structural properties of nitrogenase and shed light on previously uncharacterized states of these proteins.
", "doi": "10.7907/p232-4w49", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15265", "collection": "thesis", "collection_id": "15265", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012023-220838845", "primary_object_url": { "basename": "2023_TomaleriGP_V08.pdf", "content": "final", "filesize": 63670316, "license": "other", "mime_type": "application/pdf", "url": "/15265/2/2023_TomaleriGP_V08.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Structure and Mechanism of the Human ER Membrane Protein Complex", "author": [ { "family_name": "Pinton Tomaleri", "given_name": "Giovani", "orcid": "0000-0001-9661-6480", "clpid": "Pinton-Tomaleri-Giovani" } ], "thesis_advisor": [ { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The successful synthesis, targeting, insertion, folding, and assembly of membrane proteins into designated membranes is a crucial process in cell biology. Recent research has shed valuable light on this process through the discovery of the endoplasmic reticulum (ER) membrane protein complex (EMC) and its role in membrane protein biogenesis and quality control. As part of my Ph.D. research in the Voorhees lab, I collaborated with esteemed scientists to investigate the EMC in atomic detail. Described in this thesis is the mechanistic basis of EMC function in membrane protein biogenesis, including recent insights into its broader role beyond its well-defined insertase function. First, we determined the structure of the human EMC using single-particle cryo-electron microscopy (cryo-EM). The structure revealed that it utilizes a mechanism similar to other protein-conducting channels, which involves membrane thinning and polar intramembrane residues to transport substrate transmembrane domains from the cytosol into the membrane. The EMC structure provided the foundation for the subsequent rigorous analysis of its role in membrane protein biogenesis. Through this work, we demonstrate the molecular mechanisms involved in the EMC-dependent path a membrane protein takes, from its initial cytosolic capture by methionine-rich loops of the EMC to its eventual membrane insertion via a hydrophilic vestibule. We further show that specific polar intramembrane residues on the EMC serve as an ER \u201cselectivity filter\u201d which uses charge-repulsion properties to reject mis-targeted mitochondrial membrane proteins and maintain organelle integrity. We also demonstrate that the EMC ensures that transmembrane-spanning substrates adopt the correct topology by promoting the \u201cpositive-inside\u201d rule, which states that positively and negatively charged amino acids localize to the interior (cytoplasmic) and exterior (non-cytoplasmic) sides of membranes, respectively. Finally, our studies suggest that the EMC has a broader role beyond its well-defined insertase function. Specifically, we found that the EMC physically binds to other factors involved in membrane protein biogenesis, providing a shared interaction surface that acts as a hub to integrate signals from other pathways. Using a combination of structural and functional approaches, we identified the EMC\u2019s interaction with Nodal modulator (NOMO) complex, which is part of the multipass translocon complex and facilitate membrane protein biogenesis. Together, these results define and expand the model for membrane protein biogenesis at the ER membrane by the EMC and highlight the complex interplay between different factors in this important process. Together, these results define and expand the model for membrane protein biogenesis at the ER membrane by the EMC and highlight the complex interplay between different factors in this important process.", "doi": "10.7907/e85y-re86", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15157", "collection": "thesis", "collection_id": "15157", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05092023-163511031", "primary_object_url": { "basename": "PhD_thesis_final.pdf", "content": "final", "filesize": 56663983, "license": "other", "mime_type": "application/pdf", "url": "/15157/5/PhD_thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Cryo-ET Reveals Molecular Details of Multi-Megadalton Bacterial Protein Complexes", "author": [ { "family_name": "Dutka", "given_name": "Przemys\u0142aw", "orcid": "0000-0003-3819-1618", "clpid": "Dutka-Przemys\u0142aw" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cryo-electron tomography (cryo-ET) is a powerful method for investigating the 3D structure of intact cells, organelles, and complex protein macromolecules that cannot be crystallized or are too heterogenous for single-particle cryo-electron microscopy (cryo-EM). However, obtaining high- resolution cryo-ET structures for many biologically important targets is still a challenge. To address this challenge, cryo-ET can be combined with other methods, including X-ray crystallography, single-particle cryo-EM, structure predictions, cross-linking mass spectrometry, biochemistry, and evolutionary analysis to produce integrative models. Recently, with the development of AI-based tools such as AlphaFold2, structure prediction has played an increasingly important role in integrative modeling. The combination of cryo-ET and structure prediction in particular has provided unprecedented insights into the ultrastructure of cellular components. This thesis focuses on two bacterial multi-megadalton protein complexes which are difficult to study by classical structural biology approaches: gas vesicles (GVs) and the Legionella pneumophila Dot/Icm type IV secretion system (T4SS). GVs are gas-filled protein nanostructures that regulate the position of certain microorganisms in water and consequently their access to sunlight and nutrients. Here, we investigate the mechanical properties of GVs and reveal the molecular structure of GVs and its implication for the assembly mechanism. The Dot/Icm T4SS is a macromolecular complex formed by approximately 27 proteins, utilized by L. pneumophila to hijack the host cell's biology for its replication purposes. A nearly-complete integrative model of this complex provides crucial insights into its structural organization and its evolution from conjugation to secretion, as well as the transportation of substrates into the host cell.
", "doi": "10.7907/87jm-7v06", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14987", "collection": "thesis", "collection_id": "14987", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07252022-075028264", "primary_object_url": { "basename": "2023_Thesis_Hao-Hsuan_Hsieh_Final.pdf", "content": "final", "filesize": 29274358, "license": "other", "mime_type": "application/pdf", "url": "/14987/1/2023_Thesis_Hao-Hsuan_Hsieh_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Coordination Between Mammalian Nascent Protein Targeting and Cotranslational Chaperones", "author": [ { "family_name": "Hsieh", "given_name": "Hao-Hsuan", "orcid": "0000-0001-9629-5832", "clpid": "Hsieh-Hao-Hsuan" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein biogenesis starts with ribosome synthesizing nascent polypeptide chain. Ribosome is a major hub for multiple pathways including membrane targeting, chaperones, chemical modification, and quality control. All these pathways need to coordinate with each other spatially on the ribosomal surface and temporally within the translation elongation window. Accumulating data start to point to more intricate interaction and coordination between different pathways beyond the simple competition traditionally presumed.
\r\n\r\nIn Chapter 1, I demonstrate the coordination between a cotranslational chaperone, NAC, and the ER targeting machinery, SRP. NAC and SRP can bind to the same ribosome simultaneously despite overlapping binding sites, allowing NAC to change conformation of SRP specifically to the NC sequences. This allostery enhances the specificity of SRP-SR association, explaining the long-observed effect of NAC modulating ER targeting specificity.
\r\n\r\nIn Chapter 2, I dig deeper into the mechanism of NAC regulating SRP. Based on cryo-EM structures, NAC domain sits on top of the ribosomal tunnel exit, potentially sensing the identity of NC, and is anchored by positively charged NAC\u03b2 N-terminal tail. NAC-UBA domain is the key to recruiting SRP and coordinating the substrate handover to SRP.
\r\n\r\nIn Chapter 3, I focus on the cotranslational HSP40/HSP70 system of RAC. Ribosome binding of RAC stimulates its cochaperone activity to activate HSP70 ATP hydrolysis. Ribosome sensing by RAC is related to the NBD of HSPA14. RAC-stimulated HSP70 engagement to NC keeps it in a folding-competent unfolded state before HSP70 dissociation.
\r\n\r\nTaken together, this study advances the experimental and theoretical tools to studying cotranslational pathways associated with the mammalian ribosome and demonstrates the interesting question of coordination between cotranslational pathways.
", "doi": "10.7907/x61p-vf31", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15194", "collection": "thesis", "collection_id": "15194", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05192023-001330664", "type": "thesis", "title": "Engineering of Second-Generation Acoustic Reporter Genes", "author": [ { "family_name": "Hurt", "given_name": "Robert Cooper", "orcid": "0000-0002-4347-6901", "clpid": "Hurt-Robert-Cooper" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 \u00b5m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo.
\r\n\r\nThe goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the \u201cnature and nurture\u201d of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.
\r\n\r\nThis thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer them\u2014one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones.
\r\n\r\nThe approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.
", "doi": "10.7907/qs6v-5d67", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14626", "collection": "thesis", "collection_id": "14626", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262022-064823923", "primary_object_url": { "basename": "stefan_petrovic_2022_thesis_proofed_v03.pdf", "content": "final", "filesize": 276232032, "license": "other", "mime_type": "application/pdf", "url": "/14626/1/stefan_petrovic_2022_thesis_proofed_v03.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Structure and Function of the Human Nuclear Pore Complex", "author": [ { "family_name": "Petrovic", "given_name": "Stefan", "orcid": "0000-0002-4979-8696", "clpid": "Petrovic-Stefan" } ], "thesis_advisor": [ { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-1726-0127", "clpid": "Hoelz-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Martin", "given_name": "Andreas", "orcid": "0000-0003-0923-3284", "clpid": "Martin-Andreas" }, { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The nuclear pore complex (NPC) mediates the selective transport of macromolecules between the nucleus and the cytoplasm of the eukaryotic cell. In humans, the NPC is a ~110 MDa assembly of ~1,000 proteins, termed nucleoporins, which establishes a ~50 nm wide central transport channel that traverses both membranes of the nuclear envelope. In the central transport channel, natively unfolded nucleoporin regions rich in repeated phenylalanine-glycine (FG) motifs form a size-selective barrier that limits the diffusion of macromolecules. Active cargo transport is facilitated by dedicated mobile transport factors that exchange through the FG barrier and couple binding and unbinding of cargo to GTP hydrolysis by the small GTPase Ran. By contrast, nuclear export of mature mRNA is driven by ATP-dependent remodeling of messenger ribonucleoprotein (mRNP) at the NPC's cytoplasmic face. Whereas the mechanisms of the mobile Ran dependent transport machinery have been conceptually rationalized, the NPC's role in facilitating and modulating nucleocytoplasmic transport, including mRNA export, remains poorly understood. Addressing gaps in the understanding of the NPC's structure and function, this thesis presents comprehensive interdisciplinary analyses of the NPC's inner ring, linker scaffold, and cytoplasmic face architectures. First, we determined the structure, stoichiometry, and location of the channel nucleoporin heterotrimer that provides the bulk of the FG repeats in the central transport channel. Second, we elucidated the molecular details and topology of the network of linker nucleoporins previously shown to assemble folded scaffold nucleoporins into protomeric subcomplexes of the inner ring. Third, we characterized the composition, stoichiometry, and attachment mechanism of cytoplasmic filament nucleoporins, which present manifold Ran- and RNA-binding domains on the cytoplasmic face of the NPC. The resulting near atomic structure of the human NPC provides a rich foundation for rationalizing the dilation and constriction of the central transport channel, the nucleocytoplasmic transport of integral membrane proteins, and the spatial arrangement of cytoplasmic filament nucleoporins that are involved in mRNA export and etiologies of several human diseases.
", "doi": "10.7907/g77e-h329", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14619", "collection": "thesis", "collection_id": "14619", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252022-173757484", "primary_object_url": { "basename": "MacArdle_Siobha\u0301n_2022(2).pdf", "content": "final", "filesize": 16611071, "license": "other", "mime_type": "application/pdf", "url": "/14619/20/MacArdle_Siobha\u0301n_2022(2).pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Analytical Chemistry Investigations Toward Understanding the Mechanism of Nitrogenase from Azotobacter vinelandii and the Role of the 4Fe-4S Cluster of Dna2 from Saccharomyces cerevisiae", "author": [ { "family_name": "MacArdle", "given_name": "Siobh\u00e1n Gaustad", "orcid": "0000-0001-7843-5977", "clpid": "MacArdle-Siobh\u00e1n-Gaustad" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Agapie", "given_name": "Theodor", "orcid": "0000-0002-9692-7614", "clpid": "Agapie-T" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Iron sulfur clusters are ubiquitous metal cofactors that play a variety of roles in many enzymes important for health and the climate. The bacterial nitrogenase enzyme, which supports the growth of all organisms by converting atmospheric dinitrogen into ammonia, contains three different redox-active iron sulfur clusters that are central to its function. Dna2, found in all eukaryotes, is integral to genome maintenance and coordinates an iron sulfur cluster of unknown function. Many details of the nitrogenase mechanism are yet to be revealed and pursuits toward this goal will support human efforts to develop more sustainable solutions to nitrogen fixation, which is required for maintaining our food supply. Thorough characterization of the DNA-maintenance enzyme Dna2 will allow us to develop better technologies for cancer prevention and treatment. Development and optimization, as well as technical critique, of a variety of analytical chemistry techniques were performed toward the goal of increasing our understanding of these two important enzymes. Yeast Dna2 was successfully overexpressed and purified from E. coli and spectroscopic features of the 4Fe-4S cluster were characterized. Toward measuring the redox potential of the 4Fe-4S cluster of Dna2, the DNA-modified electrochemistry technique was evaluated leading to the discovery that the source of electrochemical signals proposed to be due to redox activity of 4Fe-4S clusters in DNA-binding proteins are actually due to the redox activity of Fe-EDTA complexes that form in the buffers of these proteins. These results will support future scientists in accurately interpreting the electrochemical signals from DNA-modified electrochemistry. The solvent isotope effect of nitrogenase reduction was investigated by measuring deuterium incorporation into nitrogenase products by GC-MS, FTIR and NMR, revealing that the enzyme exhibits modest preference for H vs. D in acetylene reduction to ethylene, but significant preference for H in the reduction of protons to dihydrogen. These results indicate that there are distinct mechanisms of H atom transfer in the reduction of these two substrates and the experimental design that we developed opens the door for a new avenue of nitrogenase research to reveal the solvent isotope effects of reduction of a variety of different substrates under different experimental conditions. Finally, a new ATPase assay using ion chromatography was developed to measure ATPase activity of the Fe protein, which provides a tool for future pursuits toward quantifying inorganic phosphate release by ATPases and led to our surprising result that the apo-form of the Fe protein is active in ATP hydrolysis.
", "doi": "10.7907/afrw-sx78", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14350", "collection": "thesis", "collection_id": "14350", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08312021-211824138", "primary_object_url": { "basename": "Holman_Elizabeth_2021_thesisfull_final.pdf", "content": "final", "filesize": 3885043, "license": "other", "mime_type": "application/pdf", "url": "/14350/1/Holman_Elizabeth_2021_thesisfull_final.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Developing Technologies for Real-Time Whole-Organism Imaging via FTIR Spectromicroscopy", "author": [ { "family_name": "Holman", "given_name": "Elizabeth Anne", "orcid": "0000-0002-5158-4689", "clpid": "Holman-Elizabeth-Anne" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "FTIR spectromicroscopy heavily resides in the domain of cell-based and tissue-based studies when focusing on its direct application to biological systems. The goal of the reported graduate research is to extend FTIR spectromicroscopy to multicellular whole-organism imaging, ideally for non-invasive, non-destructive, and label-free spatiochemical imaging of biological model Caenorhabditis elegans (C. elegans). With modern optics, detector, and light source technologies implemented at synchrotron facilities, this thesis focuses on exploring the feasibility of multicellular whole-organism imaging while identifying challenges and presenting working solutions for them.
", "doi": "10.7907/es4r-kq84", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14354", "collection": "thesis", "collection_id": "14354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09072021-015258902", "type": "thesis", "title": "Investigation of the Roles of Hopanoids in the Lifecycle of Bradyrhizobium diazoefficiens in the Context of Climate Change", "author": [ { "family_name": "Tookmanian", "given_name": "Elise M.", "clpid": "Tookmanian-Elise-M" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Ondrus", "given_name": "Alison E.", "orcid": "0000-0002-6023-3290", "clpid": "Ondrus-A-E" }, { "family_name": "Sessions", "given_name": "Alex L.", "orcid": "0000-0001-6120-2763", "clpid": "Sessions-A-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhizobia are a group of bacteria that participate in plant-growth promoting symbioses with legumes, where the bacteria supply the plant with a source of useable nitrogen. In agriculture, crop rotation capitalizes on this symbiosis by planting legumes to restore the nitrogen content of depleted soils. The effects of climate change, such as increased temperature and changing precipitation patterns, threaten the future viability of agriculture. Rhizobia exemplify the role bacteria can play to improve agriculture\u2019s resilience to climate change and prevent land degradation and food insecurity. However, in order for bacteria to realize this potential, they need to survive the challenges of climate change. In my thesis, I detail the environments that rhizobia experience throughout their lifecycle and how the soil environment will likely change as the climate changes. Then, I connect these environmental parameters, especially hypo and hyperosmolarity, to the outer membrane. The outer membrane is the first line of defense for bacteria against external assaults. Rhizobia make many changes to their outer membrane compared to commonly studied enteric bacteria. For example, the ability to synthesize hopanoids, steroid-like lipids, is overrepresented in rhizobia.
\r\n\r\nHopanoids are known to help protect bacteria against a wide range of stresses \u2013 but, surprisingly, we found that the extended hopanoid class is not required for a moderately successful symbiosis between rhizobia strain Bradyrhizobium diazoefficiens and the tropical legume Aeschynomene afraspera. The main defect was in the initiation of the symbiosis, perhaps due to motility defects in the extended hopanoid\u2014deficient mutant. As we investigated this paradox, we discovered that hopanoids are conditionally essential in B. diazoefficiens depending on the medium in which the organism is grown. Specifically, we investigated the role of hypoosmolarity and divalent cation concentration, discovering that extended hopanoids confer robustness to the physicochemical environment. This property indicates that extended hopanoids may be important in the soil environment, which is prone to osmotic variability, especially as the climate changes. This work increases our understanding of the role of the outer membrane and hopanoids in bacterial resilience which may help with engineering or selection of better crop additives in the future.
", "doi": "10.7907/h0xe-jb65", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14619", "collection": "thesis", "collection_id": "14619", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252022-173757484", "primary_object_url": { "basename": "MacArdle_Siobha\u0301n_2022(2).pdf", "content": "final", "filesize": 16611071, "license": "other", "mime_type": "application/pdf", "url": "/14619/20/MacArdle_Siobha\u0301n_2022(2).pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Analytical Chemistry Investigations Toward Understanding the Mechanism of Nitrogenase from Azotobacter vinelandii and the Role of the 4Fe-4S Cluster of Dna2 from Saccharomyces cerevisiae", "author": [ { "family_name": "MacArdle", "given_name": "Siobh\u00e1n Gaustad", "orcid": "0000-0001-7843-5977", "clpid": "MacArdle-Siobh\u00e1n-Gaustad" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Agapie", "given_name": "Theodor", "orcid": "0000-0002-9692-7614", "clpid": "Agapie-T" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Iron sulfur clusters are ubiquitous metal cofactors that play a variety of roles in many enzymes important for health and the climate. The bacterial nitrogenase enzyme, which supports the growth of all organisms by converting atmospheric dinitrogen into ammonia, contains three different redox-active iron sulfur clusters that are central to its function. Dna2, found in all eukaryotes, is integral to genome maintenance and coordinates an iron sulfur cluster of unknown function. Many details of the nitrogenase mechanism are yet to be revealed and pursuits toward this goal will support human efforts to develop more sustainable solutions to nitrogen fixation, which is required for maintaining our food supply. Thorough characterization of the DNA-maintenance enzyme Dna2 will allow us to develop better technologies for cancer prevention and treatment. Development and optimization, as well as technical critique, of a variety of analytical chemistry techniques were performed toward the goal of increasing our understanding of these two important enzymes. Yeast Dna2 was successfully overexpressed and purified from E. coli and spectroscopic features of the 4Fe-4S cluster were characterized. Toward measuring the redox potential of the 4Fe-4S cluster of Dna2, the DNA-modified electrochemistry technique was evaluated leading to the discovery that the source of electrochemical signals proposed to be due to redox activity of 4Fe-4S clusters in DNA-binding proteins are actually due to the redox activity of Fe-EDTA complexes that form in the buffers of these proteins. These results will support future scientists in accurately interpreting the electrochemical signals from DNA-modified electrochemistry. The solvent isotope effect of nitrogenase reduction was investigated by measuring deuterium incorporation into nitrogenase products by GC-MS, FTIR and NMR, revealing that the enzyme exhibits modest preference for H vs. D in acetylene reduction to ethylene, but significant preference for H in the reduction of protons to dihydrogen. These results indicate that there are distinct mechanisms of H atom transfer in the reduction of these two substrates and the experimental design that we developed opens the door for a new avenue of nitrogenase research to reveal the solvent isotope effects of reduction of a variety of different substrates under different experimental conditions. Finally, a new ATPase assay using ion chromatography was developed to measure ATPase activity of the Fe protein, which provides a tool for future pursuits toward quantifying inorganic phosphate release by ATPases and led to our surprising result that the apo-form of the Fe protein is active in ATP hydrolysis.
", "doi": "10.7907/afrw-sx78", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14592", "collection": "thesis", "collection_id": "14592", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202022-005152490", "primary_object_url": { "basename": "thesis_draft13.pdf", "content": "final", "filesize": 290818840, "license": "other", "mime_type": "application/pdf", "url": "/14592/9/thesis_draft13.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structural Basis of Antibody Recognition of Viruses", "author": [ { "family_name": "Esswein", "given_name": "Shannon Rose", "orcid": "0000-0002-5142-0190", "clpid": "Esswein-Shannon-Rose" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_chem" } ], "abstract": "The Zika epidemic in 2015-2016 and COVID-19 pandemic in 2019-2021 are the latest reminders of the enormous impact of viruses on the world. Zika, a flavivirus transmitted by mosquitos, can cause severe neurodevelopmental abnormalities including microcephaly in the newborns of the infected mothers. Vaccine design is complicated by concern that elicited antibodies may also recognize other epidemic-causing flaviviruses that share a similar envelope protein, such as dengue virus, West Nile Virus, and yellow fever virus. This cross-reactivity, if non-neutralizing, may worsen symptoms of a subsequent infection through antibody-dependent enhancement (ADE). To better understand the neutralizing antibody response and risk of ADE, we compared germline and mature antibody binding to Zika and other flaviviruses. We showed that affinity maturation of the light chain variable domain is important for strong binding of VH3-23/VK1-5 neutralizing antibodies to Zika virus envelope domain III (EDIII) and identified interactions that contribute to weak, cross-reactive binding to West Nile Virus EDIII. These findings informed our design of EDIII-conjugated mosaic nanoparticles as a pan-flavivirus vaccine candidate. Sera from immunization trials with nanoparticles displaying EDIIIs of Zika and dengue serotypes 1-4 showed cross-reactive binding to Zika, dengue 1-4, and West Nile Virus, a promising step towards the development of safe and effective flavivirus vaccines.
\r\n\r\nCoronaviruses are another group of viruses responsible for widespread morbidity and mortality, including the severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) epidemics and current SARS-CoV-2 pandemic. Given concerns regarding new SARS-CoV-2 variants and the possibility for additional zoonotic betacoronaviruses to cause future outbreaks, we investigated how the epitopes on the SARS-CoV-2 receptor binding domain (RBD) targeted by VH3-30-derived antibodies correlate with their neutralization potency and breadth of betacoronavirus recognition. Analyses showed how variations in antibody light chains and CDRH3 lengths facilitate the diverse RBD epitopes, cross-reactivity, and neutralization profiles of VH3-30 Abs, illustrating their importance for vaccine design and therapeutic antibody development.
", "doi": "10.7907/krjp-se81", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14487", "collection": "thesis", "collection_id": "14487", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02012022-191225421", "type": "thesis", "title": "Antibody Targeting of HIV-1 Env: a Structural Perspective", "author": [ { "family_name": "Abernathy", "given_name": "Morgan Elizabeth", "orcid": "0000-0001-9959-7713", "clpid": "Abernathy-Morgan-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "COVID-19" }, { "literal": "div_chem" } ], "abstract": "A key component of contemporary efforts toward a human immunodeficiency virus 1 (HIV-1) vaccine is the use of structural biology to understand the structural characteristics of antibodies elicited both from human patients and animals immunized with engineered 'immunogens,' or early vaccine candidates. This thesis will report on projects characterizing both types of antibodies against HIV-1. Chapter 1 will introduce relevant topics, including the reasons HIV-1 is particularly capable of evading the immune system in natural infection and after vaccination, the 20+ year history of unsuccessful HIV-1 vaccine large-scale efficacy trials, an introduction to broadly neutralizing antibodies (bNAbs), and a review of common strategies utilized in HIV-1 immunogen design today. Chapter 2 describes the isolation, high-resolution structural characterization, and in vitro resistance profile of a new bNAb, 1-18, that is both very broad and potent, as well as able to restrict HIV-1 escape in vivo. Chapter 3 reports the results of an epitope-focusing immunogen design and immunization experiment carried out in wild type mice, rabbits, and non-human primates where it was shown that B cells targeting the desired epitope were expanded after a single prime immunization with immunogen RC1 or a variant, RC1-4fill. Chapter 4 describes Ab1245, an off-target non-neutralizing monoclonal antibody isolated in a macaque that had been immunized with a series of sequential immunogens after the prime immunization reported in Chapter 3. The antibody structure describes a specific type of distracting response as it binds in a way that causes a large structural change in Env, resulting in the destruction of the neutralizing fusion peptide epitope. Chapter 5 is adapted from a review about how antibodies differentially recognize the viruses HIV-1, SARS-CoV-2, and Zika virus. This review serves as an introduction to the virus SARS-CoV-2, which is the topic of the final chapter, Chapter 6. In this chapter, structures of many neutralizing antibodies isolated from SARS-CoV-2 patients were used to define potentially therapeutic classes of neutralizing receptor-binding domain (RBD) antibodies based on their epitopes and binding profiles.", "doi": "10.7907/0p2b-aa45", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14483", "collection": "thesis", "collection_id": "14483", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01222022-001845303", "type": "thesis", "title": "Conformational Plasticity of HIV-1 Env and Implications for Vaccine Design", "author": [ { "family_name": "Yang", "given_name": "Zhi", "orcid": "0000-0001-8680-3784", "clpid": "Yang-Zhi" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The human immunodeficiency virus (HIV) envelope glycoprotein (Env), a (gp120/gp41)\u2083 trimer, is present on the surface of the viral envelope membrane. Env binding to the host cell receptor, CD4, and the co-receptor, CCR5 or CXCR4, triggers a cascade of Env conformational changes and structural rearrangements which ultimately leads to the viral and host cell membrane fusion, marking the initiation of a viral infection. In this work, we present findings of the conformational changes of an Env trimer from a closed, pre-fusion state to an asymmetrically open state when bound to receptor CD4 and a co-receptor mimicking antibody, E51. We showed the importance of tyrosine sulfation in gp120 binding. The EM structures also indicate the existence of Env\u2019s multiple conformational states. Based on the structural information, we modeled the order of conformations on the path to co-receptor binding and viral-host cell membrane fusion.
\r\n\r\nAs the sole viral protein present on the virion surface, the Env acts as the target for anti-HIV antibodies. Using various types of engineered Env as the immunogen, researchers made attempts to elicit anti-HIV neutralizing antibodies in animals. In this work, we identified and analyzed two neutralizing antibodies, Ab1303 and Ab1573, that target the Env CD4 binding site (CD4bs), one of the conserved epitopes on the Env gp120 surface. Using biophysical and structural methods, we described a novel recognition mechanism of these antibodies and proposed a model about the unique behavior of Env under physiological conditions. This study proved that CD4bs Abs that recognize an \"occluded open\" Env can be raised by sequential animal immunizations, thereby guiding the future immunogen design and therapeutic applications.
", "doi": "10.7907/99hp-y284", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14252", "collection": "thesis", "collection_id": "14252", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072021-073626662", "type": "thesis", "title": "Investigating the Catalytic Mechanisms of Bio-degrading Copper Proteins: Multi-copper Oxidases (MCOs) and Lytic Polysaccharide Monooxygenases (LPMOs)", "author": [ { "family_name": "Shin", "given_name": "Jieun", "orcid": "0000-0003-3817-6282", "clpid": "Shin-Jieun" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "orcid": "0000-0002-4453-9716", "clpid": "Winkler-J-R" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Lignin and cellulose comprise a large portion of the renewable biomass on Earth. However, substantially due to laborious course of processing, the conversion efficiency of these biomaterials to accessible biofuel is very low. Therefore, effective depolymerization and utilization of these biopolymers are requirements for environmentally friendly and sustainable energy development. In the hope of finding solutions to these biomass utilization challenges, there have been growing interests in using biodegrading metalloenzymes as active biocatalysts. However, there still remain many questions regarding mechanistic details of enzyme catalysis and effective application of these enzymes. This thesis focuses on investigating the redox chemistry involved in the catalytic mechanisms of two main lignin- and cellulose- degrading copper enzymes: multicopper oxidases (MCOs) and lytic polysaccharide monooxygenases (LPMOs).
\r\n\r\nMCOs are capable of aerobic oxidation of lignin as their primary function, but the nature of their substrate variability also allows the oxidation of not only diverse high potential organic and inorganic complexes, but also earth abundant divalent metal ions such as manganese. LPMOs, on the other hand, enable the cleavage of glycosidic bonds in recalcitrant insoluble cellulosic substances, which are not degradable by other hydrolytic enzymes such as endoglucanases and cellulobiohydrolases.
\r\n\r\nIt is remarkable that nature has created such versatile enzymes with specific active site metals and redox-active amino acids involved in electron transfer, which contribute to substrate oxidation as well as enzyme survival against oxidative damage during catalysis. By gaining a deeper understanding of how these enzymes work, we could greatly enhance current usage efficiencies and develop more energy-efficient biocatalysts.
\r\n\r\nChapter I gives an introduction to biological coppers, two groups of bio-degrading copper enzymes: multicopper oxidases (MCOs) and lytic polysaccharide monooxygenases (LPMOs), and the role of redox-active amino acids in electron transfer and enzyme catalysis. For the MCO work, a thermophilic laccase (Tth-lac) from Thermus thermophilus HB27 and a CotA laccase (CotA-lac) from Bacillus Subtilis were studied. For the LPMO work, two cellulose active LPMOs (ScLPMO10B and ScLPMO10C) and a chitin active LPMO (BlLPMO10A) were studied.
\r\n\r\nChapter II describes thermodynamic aspects of Tth-lac catalysis. The temperature dependence of the formal potential of type I copper (CuT1) in Tth-lac is reported, and the interplay between many competing dynamic and thermodynamic factors which results in thermostability and activity of Tth-lac is discussed.
\r\n\r\nChapter III reports the electron transfer (ET) kinetics data obtained with Tth-lac using the transient absorption spectroscopy. The results of photochemical electron/hole transfer studies indicate that the chains of Trp and Tyr can participate in electron transfer through Tth-lac, which could potentially have a role in enzyme catalysis as well.
\r\n\r\nChapter IV discusses the protective role of a Trp/Tyr pair positioned close to the trinuclear copper cluster (TNC) in Tth-lac. It is indeed remarkable that laccases are capable of utilizing the power of oxygen to catalyze the oxidation of diverse high-potential substrates. But, as a tradeoff, the utilization of dioxygen can make the enzyme highly susceptible to oxidative damage. Chapter IV provides supporting evidence that led us to conclude that the TNC-proximal Trp/Tyr pair functions as an internal antioxidant for prolonging the enzyme lifetime.
\r\n\r\nChapter V describes investigations on the factors that affect MCO catalysis, which include the potentials of the active site coppers, possible reactive intermediates, and common structural motifs. Based on the structural homology between Tth-lac and CotA-lac, some preliminary work done on CotA-lac is also reported.
\r\n\r\nChapter VI outlines the work on LPMOs. After the successful expression and purification of ScLPMO10B, ScLPMO10B and BlLPMO10A, standard activity assays were done with insoluble cellulose and chitin substrates to confirm the enzyme activity. The results are compared with that from the photo-degradation experiments to investigate if the photochemically generated Cu(III) species are active intermediates in LPMO catalysis.
\r\n\r\nChapter VII reports the results on bioinformatics analysis on the distribution of vicinal amino acids in different enzyme classes. This study was to examine the biological significance of amino acid pairs and clusters existing in many different enzyme classes, with vicinal surface tyrosines in CotA-lac as an underlying motivation behind the work.
\r\n\r\nThis thesis demonstrates that MCOs and LPMOs are truly versatile enzymes which can oxidize such diverse refractory substrates, and there could be multiple pathways that the enzymes achieve this task. As shown so far, not only the active site metals but also the chain of redox-active amino acids as well as metal coordinating residues can contribute to enzyme catalysis.
", "doi": "10.7907/m5n0-ck53", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13870", "collection": "thesis", "collection_id": "13870", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09082020-225341960", "primary_object_url": { "basename": "Proofread_v_Threatt-thesis-compiled.pdf", "content": "final", "filesize": 11152640, "license": "other", "mime_type": "application/pdf", "url": "/13870/1/Proofread_v_Threatt-thesis-compiled.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "In Vivo Activity of Rhodium Metalloinsertors and Exploration of Drug Delivery Systems", "author": [ { "family_name": "Threatt", "given_name": "Stephanie Denise", "orcid": "0000-0002-2303-2166", "clpid": "Threatt-Stephanie-Denise" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Synold", "given_name": "Timothy", "orcid": "0000-0002-4075-2544", "clpid": "Synold-Timothy" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhodium metalloinsertors are octahedral complexes developed to selectively target the mismatches and insertions/deletions (indels) that result from mismatch repair (MMR) deficient cancers. By incorporating particularly wide, aromatic, inserting ligands, these complexes are able to detect thermodynamically destabilized mismatch sites via a binding mode known as metalloinsertion, in which the inserting ligand binds DNA via the minor groove and results in ejection of the destabilized mismatched base pair. In vitro analyses of metalloinsertors have found that these complexes are selectively cytotoxic towards MMR-deficient cancer cells compared to MMR-proficient cells. Furthermore, the newest family of Rh-O metalloinsertors, which includes [Rh(phen)(chrysi)(PPO)]\u00b2\u207a (Rh-PPO), displays preferential cytotoxicities in the nanomolar range, which is significantly more potent than first generation metalloinsertors and many standard of care chemotherapeutics. Given the high level of potency and selectivity of Rh-O metalloinsertors, further clinical development of these complexes has been pursued.
\r\n\r\nHere, we present the first preclinical mouse evaluation of a rhodium metalloinsertor as an anticancer agent. The Rh-O metalloinsertor Rh-PPO was evaluated in the HCT116 colorectal cancer xenograft tumor model alongside saline and oxaliplatin controls. Intraperitoneal studies with Rh-PPO showed significant decreases in tumor volumes over time and final tumor weights, indicating Rh-PPO has notable anticancer activity. Additionally, Rh-PPO treatment resulted in a noteworthy increase in the length of mouse survival that was on par with the FDA approved chemotherapeutic oxaliplatin. Pharmacokinetic analyses revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Importantly, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, which points to a need for more selective delivery methods in order to further metalloinsertor development.
\r\n\r\nIn order to target cancerous cells with still higher selectivity, routes to metalloinsertor antibody drug conjugate (ADC) designs were explored. By attaching Rh-O metalloinsertors to an antibody specific to cancer-associated antigens, our complexes may become even more specifically directed to induce selective cytotoxicity in diseased cells. Three ADC drug linkers that incorporate maleimide groups into the N^O coordinating ligand of a Rh-O metalloinsertor were designed, synthesized, and characterized. These complexes were evaluated for their cellular potency and selectivity toward MMR-deficient cancer cells. Studies revealed that functionalization of the hydroxyl-containing ancillary ligand resulted in decreased potency and abolished preferential cytotoxicity, contrary to previous studies that assessed modifications of this ligand.
\r\n\r\nLiposomal formulations of Rh-PPO were also explored to further target metalloinsertors to malignant cells. Liposomal drug encapsulations have a demonstrated ability to decrease systemic toxicity and increase tumor drug uptake; therefore, the biological activity of Rh-PPO liposomal formulations was explored. Four distinct Rh-PPO liposome formation methods were developed and the resulting liposomes were assessed for their encapsulation efficiency, cellular toxicity, and stability. Remote loaded Rh-PPO liposomes were found to display the most promising chemical and biological characteristics, although additional optimization of encapsulation procedures is necessary for further preclinical evaluation of this metalloinsertor drug delivery approach.
\r\n\r\nAs metalloinsertors continue preclinical assessment and development, a greater understanding of their mechanism of action is imperative. Biological studies with Rh-PPO and the fluorescent analogue RhPPO-Cy3 have shown that DNA damage from metalloinsertor treatment involves the formation of DNA double strand breaks near metalloinsertor-mismatch binding sites. Furthermore, the DNA damage response, including recruitment of pH2AX and Rad51 proteins, becomes activated in response to Rh-PPO treatment. In order to further elucidate the unique mechanism of action of Rh-O metalloinsertors, which involves both metalloinsertor enantiomers binding to DNA mismatches and displaying biological activity, structural studies are ongoing. X-ray crystallography and microelectron diffraction (microED) techniques have been used in attempts to obtain a high resolution structure of Rh-O metalloinsertors bound to DNA mismatch sites. Gaining these structural insights will be critical to understanding the increased cytotoxic selectivity and uniquely high potency of these second generation metalloinsertor complexes.
\r\n\r\nThe experiments detailed in this thesis have advanced the preclinical development of rhodium metalloinsertors. The ability of Rh-O metalloinsertors to decrease tumor growth in vivo has been established. Additionally, liposomal and ADC metalloinsertor drug formulations have been pursued as drug delivery systems, and the biological mechanisms relevant to metalloinsertor activity have been analyzed. Additional efforts to study rhodium metalloinsertors will continue to advance these promising chemotherapeutics as novel, targeted treatments for MMR-deficient cancers.
", "doi": "10.7907/dmqv-ed54", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13764", "collection": "thesis", "collection_id": "13764", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012020-160106726", "primary_object_url": { "basename": "200601_thesis_final.pdf", "content": "final", "filesize": 43126054, "license": "other", "mime_type": "application/pdf", "url": "/13764/9/200601_thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structure, Function, and Application of Bacterial ABC Transporters", "author": [ { "family_name": "Fan", "given_name": "Chengcheng", "orcid": "0000-0003-4213-5758", "clpid": "Fan-Chengcheng" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single particle cryo-EM in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione binding site and the associated substrate binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provides an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide binding domains in the inward-facing conformation.
\r\n\r\nThe ATPase kinetic data was fit to a non-essential activator model with expansion to two substrate binding sites. While the structural data suggests that MgATP and GSSG bind to distinct states, outward- and inward-facing conformations, respectively, and hence might be expected to exhibit negative cooperativity, the kinetic data support a more complex interplay and also the importance of lipid molecule presence. How GSSG binding stimulates ATPase activity remains an open question and highlights the importance of the still elusive ternary complex with both MgATP and GSSG bound to NaAtm1.
\r\n\r\nBesides the structural and functional characterizations of the ABC exporter, NaAtm1, we additionally determined crystal structures of the repurposed periplasmic binding protein (PBP) from the ABC importer system. These PBPs are designed with circularly permutated GFP to act as biosensors to sense the concentrations of smoking cessation drugs and neurotransmitters under cellular conditions. The crystal structures determined for the nicotine and acetylcholine biosensors not only revealed the key residues in ligand binding, but also demonstrated similar ligand induced conformational changes as seen in other PBPs by following the Venus-flytrap mechanism.
", "doi": "10.7907/5t65-0047", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13668", "collection": "thesis", "collection_id": "13668", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04062020-155127311", "primary_object_url": { "basename": "LJS_Thesis.pdf", "content": "final", "filesize": 10864036, "license": "other", "mime_type": "application/pdf", "url": "/13668/39/LJS_Thesis.pdf", "version": "v15.0.0" }, "type": "thesis", "title": "Electronic Structures of Perfunctionalized Dodecaborate Clusters", "author": [ { "family_name": "Schwan", "given_name": "Lars Josef", "orcid": "0000-0002-1086-6698", "clpid": "Schwan-Lars-Josef" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Dodecahydro-closo-dodecaborate is only stable as a dianionic closed-shell species, however, some alkyloxy- and aryloxy-perfunctionalized dodecaborate clusters ([B12(OR)12], R = alkyl or aryl) can be isolated in hypoelectronic hypocloso-dodecaborate(1-) and hypercloso-dodecaborate(0) states. These hypoelectronic clusters display strong visible absorption bands and highly reversible redox behavior, which have inspired applications in photochemistry, charge-storage and as dopants in conducting polymers. Chapter 1 provides a historic overview of dodecaborate research with particular focus on the development and applications of hypoelectronic clusters. Chapter 2 summarizes our spectroscopic investigtion of the photochemistry and photophysical properties of aryloxy-perfunctionalized hypercloso-dodecaborate(0) clusters. Obtaining reliable photophysical data proved exceedingly difficult due to formation of reduced hypocloso-dodecaborate(1-) species through solvent photooxidation, disproportionation and solvent-cluster interactions. A detailed discussion on these issues is presented, along with the final luminescence data collected for hypercloso-dodecaborate(0) and hypocloso-dodecaborate(1-) clusters. In Chapter 3, we present evidence indicating that certain alkyloxy-perfunctionalized dodecaborate clusters can be further oxidized to a cationic state. Electrochemical and spectroelectrochemical characterization indicate reversible conversion between the dodecaborate(0) and dodecaborate(1+) state. The results are further corroborated by EPR studies on dodecaborate(1+) clusters in-situ generated using the strong oxidant tris(2,4-dibromophenyl)ammoniumyl hexachloroantimonate. In chapter 4, we present Q-band pulsed EPR results that give a quantitative measure of the spin distribution of both hypercloso-dodecaborate(1-) and super-oxidized dodecaborate(1+) clusters. This is to our knowledge the first time pulsed EPR techniques have been applied to hypoelectronic dodecaborate clusters. The EPR data indicate that the frontier orbitals of hypoelectronic dodecaborate clusters are confined to the cluster core and delocalized evenly across the B12 pseudo-icosahedron. Furthermore, we provide UV\u2013vis\u2013NIR evidence indicating that the visible and NIR electronic transitions of these clusters occur between orbitals that are largely confined to the cluster core. Chapter 5 summarizes our results and discusses future directions.", "doi": "10.7907/jf75-8k37", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13800", "collection": "thesis", "collection_id": "13800", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082020-163907557", "primary_object_url": { "basename": "Silva_RebekahMB_2020.pdf", "content": "final", "filesize": 21105463, "license": "other", "mime_type": "application/pdf", "url": "/13800/2/Silva_RebekahMB_2020.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Attributes of the [4Fe4S] Cofactor Coordinated by UvrC, a DNA Repair Enzyme", "author": [ { "family_name": "Silva", "given_name": "Rebekah Miriam Brawer", "orcid": "0000-0002-9144-4939", "clpid": "Silva-Rebekah-Miriam-Brawer" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein-bound iron sulfur clusters are critical in cells and allow proteins to carry out many essential functions as electron carriers, catalysts for challenging organic reactions, and sensors of cellular environments. A wide range of protein families are known to coordinate iron sulfur clusters, and a growing category includes proteins involved in maintenance of the genome. Within the last three decades, iron sulfur clusters have been demonstrated to be important for enzymes that function in DNA repair, DNA replication, and transcription pathways. To date, iron sulfur clusters in the cubane [4Fe4S] geometry with all cysteine ligands have been exclusively reported for DNA repair and replication enzymes. In contrast to enzymes where the cofactor is necessary for active site chemistry or directly-linked to protein function, the [4Fe4S] cluster in the overwhelming majority of repair and replication enzymes is not involved in the catalytic modification of DNA substrates. Rather, the role of the cofactor appears to vary in function from protein to protein, and has been demonstrated to be important for protein stability, in the assembly of multisubunit proteins, and for substrate recognition, among other roles. Through investigations of the redox chemistry of the cofactor, our group has found that these enzymes participate in DNA-mediated charge transport chemistry, the process through which electrons rapidly migrate through well-stacked, duplex DNA. Long-range, DNA-mediated redox signaling provides a means of rapid communication among DNA-processing proteins for organizing repair and replication activities across the nucleus.
\r\n\r\nNotably, the first observations of the [4Fe4S] cofactor associated with repair and replications enzymes has consistently occurred well after the first biochemical studies of these enzymes. In some cases, the demonstration of a [4Fe4S] center has taken place decades later after initial work. Some proteins have required use of anaerobic methods in order to detect the cofactor, perhaps explaining why in some cases the metal center had eluded observation. Analysis of protein sequences might be expected to help accelerate identification of new iron sulfur centers in repair and replication enzymes. However, even with the abundance of sequencing data available in the post-genomic era, prediction of a metal center based on sequences alone has been challenging. This is in large part because the spacing of the coordinating cysteine residues can be quite irregular, leading to a weak bioinformatic signature.
\r\n\r\nIdentifying proteins with overlooked [4Fe4S] cofactors poses an exciting challenge, and there are some elegant examples in the literature where data from genetics assays has been used in combination with careful sequence analysis to predict and discover iron sulfur centers in repair and replication enzymes. Described here is the evolution of our studies on one well-known repair enzyme from Escherichia coli, UvrC. UvrC is part of the nucleotide excision repair pathway in the Bacteria domain which is responsible for addressing the wide class of bulky, helix-distorting lesions that can form after exposure to sources such as ultraviolet light, cigarette smoke, chemotherapeutics, and protein-DNA crosslinks. UvrC, an excision nuclease with two distinct active sites that incise the phosphodiester backbone on either side of the site of damage, has been historically challenging to study. Given how essential UvrC is in repairing damaged substrates, new insight has been greatly needed.
\r\n\r\nThrough integration of several key reports from the literature regarding the sequence of UvrC and evidence that pointed to a cofactor from genetics assays, our group predicted that UvrC is a [4Fe4S] protein. Development of a new overexpression system and an anaerobic purification method allowed for isolation of UvrC in holo form. We used spectroscopic techniques to confirm that the cluster type was [4Fe4S], and a combination of spectroscopy and chromatography to demonstrate that the UvrC-bound cofactor is susceptible to oxidative degradation. We also found that loss of the cofactor, either through aerobic degradation or mutation of coordinating cysteines, is associated with aggregation of apoprotein. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 \u00b1 20 nM and 80 \u00b1 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with DNA-bound midpoint potential of 90 mV vs. NHE.
\r\n\r\nThe work detailed in this dissertation has highlighted how critical the [4Fe4S] center is for UvrC, where the cofactor has been implicated in protein stabilization, substrate binding, and redox signaling on DNA. Handling an apo form of UvrC may have led to the previous challenges catalogued by researchers. Through the development of entirely new methods to study UvrC under anaerobic conditions, many opportunities are now available to study UvrC and the NER pathway anew in vitro and in vivo. Such work will contribute additional insight on how iron sulfur clusters are essential for enzymes that maintain genomic integrity.
\r\n\r\n", "doi": "10.7907/r0j6-jk09", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13601", "collection": "thesis", "collection_id": "13601", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092019-141850032", "type": "thesis", "title": "Visualizing Small Proteins with the cryoEM Platform and The Structure of the Vibrio cholerae Type IV Competence Pilus Secretin PilQ", "author": [ { "family_name": "Weaver", "given_name": "Sara Jean", "orcid": "0000-0001-7753-6215", "clpid": "Weaver-Sara-Jean" } ], "thesis_advisor": [ { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Voorhees", "given_name": "Rebecca M", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 \u00c5 resolution overall, with 5-8 \u00c5 resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.
\r\n\r\nNatural competence is the process by which bacteria take up genetic material from their environment and integrate it into their genome using homologous recombination. In Vibrio cholerae, the Type IV competence pilus is thought to mediate DNA uptake by binding DNA and retracting back toward the cell. How the DNA enters the periplasm is unclear. One hypothesis suggests that the DNA-bound Type IV competence pilus retracts completely so that the DNA would pass through the outer membrane secretin pore (PilQ). PilQ is a 870 kDa outer membrane pore with C14 symmetry. Here, we purify the V. cholerae PilQ secretin from V. cholerae cells in amphipols for single particle cryogenic electron microscopy (cryoEM). We solve the structure to 3.0 \u00c5 and provide insight on the channel DNA may traverse through during uptake.
", "doi": "10.7907/9B9V-PK08", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13747", "collection": "thesis", "collection_id": "13747", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312020-153151158", "type": "thesis", "title": "Pezo-1 Function in Caenorhabditis elegans", "author": [ { "family_name": "Brugman", "given_name": "Katherine Irene", "orcid": "0000-0003-2625-2903", "clpid": "Brugman-Katherine-Irene" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The piezo class of mechanosensative ion channels is a recently discovered class of cation channels with orthologs found in every phylogenetic clade aside from yeast and bacteria. THey are large channels, both in gene length and in overall diameter, with the diameter of the human PIEZO2 protein measuring in at 280 \u00c5 in its full homotrimeric form. In addition, like many similar mechanosensitive channels, such as the DEG/ENaC channels, TRP channels, and TREK/TRAAK channels, they have been linked to a number of different functions within drosophila, zebrafish, and mice, including light touch, nociception, blood cell volume regulation, vascular development, and neuropathic pain. Structurally, this channel is intriguing as it possesses no previously categorized structural motifs and is organized into a central pore with a cap, surrounded by three \"propeller blade\" regions that are theorized to anchor the channel to the membrane and control gating through hydrophobic mismatch based on membrane curvature.
\r\n\r\nThe C. elegans piezo, pezo-1, has not yet been fully characterized, even though a crystal structure of part of this particular piezo was used to assist in the resolution of the first set of cryo-EM images. Here, I generated a number of GFP transcriptional fusions of non-coding potential promoter regions to track the expression of the pezo-1 gene in C. elegans, using these expression patterns to design further experiments. From these expression patterns, I identified expression in a number of neurons of the C. elegans male tail, the primary mating apparatus of the male, and identified these neurons as key neurons as relating to mating. In particular, I identified neurons HOB, PCB, PCC and various ray neurons as potential candidates, which are ciliated neurons theorized to have mechanosensitive properties. In addition, I also identified expression in the vulva muscle and spermatheca of the hermaphrodite, both theorized to be involved in ovulation and egg-laying processes.
\r\n\r\nFrom there, I designed CRISPR/Cas9 mutants with defects in pezo-1 in order to investigate the potential link between the pezo-1 expression in those neurons and mating behavior via a mating assay. Similarly, I devised a fecundity assay to investigate the link between pezo-1 expression in ovulation organs and progeny survival. I have discovered that pezo-1 has function in both of these areas, with pezo-1 mutant males demonstrating discrete mating defects that correlate with the expression pattern seen from the GFP transcriptional fusion mutants and with pezo-1 mutant hermaphrodites having much smaller brood sizes than wildtype hermaphrodites. However, I have also discovered that the processes this mechanotransducer is involved in are also more complex than I originally believed, as I discovered that pezo-1 appears to interact with another mechanotransducer, trp-4, illuminating some potentially novel pathway considerations for how these channels overlap in function.
", "doi": "10.7907/fz4z-c850", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11282", "collection": "thesis", "collection_id": "11282", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11262018-103442842", "primary_object_url": { "basename": "AZ_thesis_FINAL.pdf", "content": "final", "filesize": 12244069, "license": "other", "mime_type": "application/pdf", "url": "/11282/1/AZ_thesis_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways", "author": [ { "family_name": "Zhou", "given_name": "Andy", "orcid": "0000-0003-3383-0855", "clpid": "Zhou-Andy" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The 4Fe4S cluster has been identified in various DNA-processing proteins spanning a variety of biological functions and all domains of life. Recently, a novel functional role for the cluster has been identified for proteins in DNA repair and replication as a redox switch for DNA binding. Human DNA primase utilizes this redox switch to coordinate primer handoff in replication. The enzymatic activity of DNA polymerase \u03b4 is tuned by the redox-switch, allowing for a fast and reversible regulation of replication in response to oxidative stress. In all cases, the redox of the 4Fe4S cluster is achieved through DNA-mediated charge transport (CT), the ability for DNA to carry charge through its \u03c0-stack. Due to the reliance of this phenomena on the \u03c0-stacking of the nitrogenous bases, DNA CT is sensitive to DNA lesions and mismatches and can proceed over long molecular distances if the DNA is well-stacked. Given this powerful biological phenomena, new inter-protein signaling interactions have been identified with important downstream consequences for genome fidelity. Here, we investigate the ways DNA-mediated charge transport between DNA processing enzymes results in efficient DNA repair or prevention of DNA-damage.
\r\n\r\nFirst, we investigated Dps, a bacterial ferritin that protects DNA from oxidative stress and implicated in bacterial survival and virulence. Dps iron sites can scavenge diffusing oxidants directly but additionally electrons and electron holes can be rapidly transported through the base-pair \u03c0-stack though DNA CT, thus providing an additional mechanism of genome protection by Dps. Using X-band EPR, we monitored formation of mononuclear high-spin Fe(III) sites of low symmetry as a gauge of effective Dps protection via oxidation of its iron sites. Using poly(dGdC)2 or poly(dAdT)2 DNA, we uncovered the dependence of DNA protection by Dps to the formation of guanine radical intermediates. Oxidation of Dps iron sites depended on the presence of the W52 residue. Point mutations of W52 revealed its involvement in an electron transfer (ET) pathway for the oxidation of the Dps iron sites. Finally, we investigated the in vivo consequences of the Dps W52 residue by complementing knockout Dps E.coli with plasmids expressing WT, W52A, or W52Y Dps and applying oxidative stress to the cells through hydrogen peroxide treatment. These assays further demonstrated the ability of Dps to protect the E.coli genome from harmful oxidants DNA-mediated electron transfer processes.
\r\n\r\nSecond, we assessed the redox properties of EndoIII and MutY, two base excision repair glycosylases containing 4Fe4S clusters, in the presence and absence of DNA. Previous work has shown these proteins to have a midpoint redox potential around 80mV vs. NHE when bound to DNA with a positive shift in potential in the absence of DNA. However, electrochemical details that define this midpoint potential have not been uncovered. Using a pyrolytic graphite edge electrode, we measured the midopoint potential of point mutations of EndoIII where point charges are flipped near the cluster (K208E, Y205H, and E200K) in the absence of DNA. Our measurements suggest that a change in a single point charge is not enough to shift the 4Fe4S cluster midpoint potential dramatically. Addition of a poly-L-glutamate polyanion introduced a slight negative shift (~20mV), but with the introduction of DNA a large negative shift was observed (70mV). Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the 4Fe4S cluster, helping to explain why all DNA binding proteins with 4Fe4S clusters studied to date have similar DNA-bound potentials.
\r\n\r\nWith these similar DNA-bound potentials, inter-protein redox signaling should occur. Previous works have demonstrated DNA-mediated redox signaling such as EndoIII signaling to DinG helicase, involved in R-loop maturation, increasing cellular survival by resolving deleterious R-loops. Additionally, different cluster- containing repair proteins of different functions and domains of life have been shown using atomic force microscopy (AFM) to localize to DNA mismatches through a redox switch for DNA-binding affinity. Given a DNA-mediated redox signaling system to scan the genome for lesions, the expression levels of these proteins may play a role in defining the scanning efficiency. We identified that the EndoIII E.coli knockout strain was sensitive to UV irradiation. This implies that EndoIII assists the nucleotide excision repair (NER) pathway via DNA-mediated redox signaling. However, knockout of MutY, another 4Fe4S glycosylase, does not impart the same UV sensitivity, and thus suggests key differences between MutY and EndoIII that define effective DNA-mediated redox signaling. Thus, the effect of protein expression level on the efficiency of DNA-mediated redox signaling was investigated using inducible protein expression of EndoIII to rescue UV-sensitivity. Using both plasmid-based and genome integrated constructs, we uncovered that low amounts of EndoIII expression were enough to rescue the growth defect, and overexpression of WT EndoIII leads to a greater defect caused by excess non-specific enzyme activity. These findings further informed investigation of this unique protein signaling interaction between EndoIII and NER protein UvrC.
\r\n\r\nWith proper EndoIII rescue plasmids, we further characterized the DNA- mediated redox signaling interaction between EndoIII and UvrC. Using UV-irradiation of genetic knockout strains and growth curve analysis, we demonstrate that EndoIII expression is essential for efficient repair of UV-induced DNA lesions, as measured through quantitative changes in growth lag-time when wild-type or mutant EndoIII is present in the cell. Electrochemical analysis of EndoIII point mutants quantify the DNA-CT inefficiencies that lead to the observed phenotypes. EndoIII, a BER repair protein, assists the NER pathway in the repair of UV-induced DNA lesions via DNA-mediated redox signaling. These results give evidence of a new signaling crosstalk between two distinct DNA repair pathways.
", "doi": "10.7907/G7NF-S349", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11325", "collection": "thesis", "collection_id": "11325", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12212018-140003184", "type": "thesis", "title": "Exploring the Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Boyle", "given_name": "Kelsey Melinda", "orcid": "0000-0002-6728-8403", "clpid": "Boyle-Kelsey-Melinda" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhodium metalloinsertors are a unique family of potential anticancer agents that have been show to bind selectively to thermodynamically destabilized DNA base pair mismatches, abasic sites, and insertions/deletions (indels) in vitro. These metalloinsertors are also able to target mismatches in cells: metalloinsertors preferentially kill mismatch repair (MMR)-deficient cancer cells, which have a relative abundance of uncorrected DNA mismatches and indels, over MMR-proficient cells, which can repair these lesions. As such, these complexes have shown great promise as a potential treatment strategy for MMR-deficient cancers, which are often resistant to classic chemotherapies.
\r\n\r\nRecently, a new class of metalloinsertors that bear a rhodium-oxygen bond was synthesized and shown to have remarkable potency and selectivity towards MMR-deficient cells. We have discovered many key differences between first generation metalloinsertors and these new Rh-O metalloinsertors: (1) the MMR-selectivity of first generation metalloinsertors is heavily influenced by ancillary ligand bulk and lipophilicity, whereas the MMR-selectivity of Rh-O metalloinsertors is strong regardless of ancillary ligand properties, (2) first generation metalloinsertors have toxicities in the micromolar range while Rh-O metalloinsertors have toxicities in the nanomolar range, and (3) first generation metalloinsertors can only bind DNA via the \u0394-enantiomer while Rh-O metalloinsertors can bind DNA via both the \u0394- and \u039b-enantiomers. Excitingly, the improved potency and selectivity of these \"Rh-O\" metalloinsertors brings them into a realm of clinical relevance.
\r\n\r\nHere we examine the basis for the improved potency and selectivity of these new Rh-O metalloinsertors. A family of six Rh-O metalloinsertors that vary in the steric bulk and lipophilicity of an ancillary ligand was synthesized and characterized. Regardless of ancillary ligand identity, these Rh-O metalloinsertors exhibit nanomolar or low-micromolar toxicities and all preferentially target MMR-deficient cancer cells over MMR-proficient cells. Notably, the off-target accumulation of these metalloinsertors in mitochondria is very low. This cellular distribution is in stark contrast with first generation metalloinsertors in which increased ligand lipophilicity led to increased mitochondrial uptake and ultimately non-selective mitochondrial-mediated cell death. We believe robust selectivity of these complexes is retained in part due to their low off-target accumulation in the mitochondria, which is further complemented by the low dosing requirements of these potent therapeutic agents.
\r\n\r\nOur studies also suggest the high potency of these complexes may be due to a difference in DNA-binding abilities, which is supported by observed differences in which enantiomers can bind to DNA mismatches, differences in ligand buckling at physiological pH, and lipophilicity of the therapeutics, with Rh-O metalloinsertors being dramatically more lipophilic than their first generation counterparts. To better understand the structural basis for this increased potency, crystallographic experiments are underway. A first generation metalloinsertor was previously crystallized with mismatched DNA, and the structure was pivotal in identifying the DNA binding mode of metalloinsertion. Using similar methods, we are working to produce a high-resolution crystal structure of an Rh-O metalloinsertor with mismatched DNA in order to gain structural insights into the increased potency of these new complexes. A significant difference in DNA binding could result in different biological activation of proteins and overall higher potency of these Rh-O metalloinsertors.
\r\n\r\nFinally, as metalloinsertors are moved towards pre-clinical study, understanding their biological activity in diverse cell culture experiments is essential. We examined a metalloinsertor and the FDA approved chemotherapeutic agent cisplatin in 27 diverse colorectal cancer cell lines. The comparison of these drugs revealed the metalloinsertor to be on average five times more potent than cisplatin in this panel. The potency of the metalloinsertor in different cell lines spanned nearly three orders of magnitude and correlated with whole-cell uptake of rhodium. Additionally, a fluorescent metalloinsertor conjugate was used to quantify the number of lesions in DNA that could be targeted by metalloinsertion, a result that correlated well with the potency of a metalloinsertor across several cell lines, consistent with DNA mismatches as the effective biological target of the metalloinsertor.
\r\n\r\nThe experiments described within this thesis have allowed us to gain a better understanding of the biological activity of rhodium metalloinsertors. We have established that Rh-O metalloinsertors are distinct from first generation metalloinsertors, and that these new metalloinsertors can serve as highly tunable, potent, and mismatch-selective anticancer agents. Furthermore, this potency is observed across diverse cell lines and has been shown to correlate with the number of genomic DNA lesions that can be bound by metalloinsertion. The unique biological activity of these complexes makes them ideal candidates for the treatment of MMR-deficient cancers, and the potency and tunability of Rh-O metalloinsertors will allow for the development of previously unattainable diagnostic and therapeutic tools for MMR-deficiencies.
", "doi": "10.7907/1KNM-Y111", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11482", "collection": "thesis", "collection_id": "11482", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04222019-175445561", "type": "thesis", "title": "The Many Roles of the Nitrogenase Iron Protein", "author": [ { "family_name": "Wenke", "given_name": "Belinda B.", "orcid": "0000-0003-3214-6197", "clpid": "Wenke-Belinda-B" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nitrogenase is the only known enzyme capable of reducing atmospheric nitrogen (N2) into ammonia (NH3) for incorporation into cellular material. N2 reduction by nitrogenase is accomplished by sequential electron transfer between two component proteins: the substrate reductase (the MoFe-protein), and a specialized low-potential electron donor (the Fe-protein). The MoFe-protein contains the active site for nitrogen reduction, the FeMocofactor (FeMo-co). During nitrogen reduction, each Fe-protein dimer docks onto the MoFe-protein, transferring electrons to an intermediate cluster (P-cluster), and ultimately to the FeMo-co.
\r\n\r\nStrikingly, the Fe-protein has another critical role in nitrogen fixation. The Fe-protein is required for the biosynthesis of the two unique metalloclusters of the MoFe-protein: the Pcluster [8Fe:7S] and the active site FeMo-co ([Mo:7Fe:9S:C]-R-homocitrate) cluster. During FeMo-co-cluster maturation, the Fe-protein forms a complex with NifEN, a scaffolding protein homologous to the MoFe-protein, catalyzing the final step in the FeMoco biosynthesis. Studies indicate that the Fe-protein catalyzes insertion of molybdenum and R-homocitrate into an all-iron FeMo-co precursor in a reductant and nucleotide dependent manner. The remaining questions about the cellular functions of the Fe-protein include how the Fe-protein interacts with other maturation proteins in distinct (or similar) ways compared to the MoFe-protein, and how the Fe-protein contributes to the activation and insertion of molybdenum into the FeMo-co.
", "doi": "10.7907/8RB1-HC30", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11485", "collection": "thesis", "collection_id": "11485", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04232019-210733826", "type": "thesis", "title": "Subunit Selective Degradation of WIZ, a Lenalidomide- and Pomalidomide-Dependent Substrate of E3 Ubiquitin Ligase CRL4CRBN", "author": [ { "family_name": "Yu", "given_name": "Helen", "orcid": "0000-0001-9881-8414", "clpid": "Yu-Helen" } ], "thesis_advisor": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation is focused on identifying novel targets of immunomodulatory(IMiD) drugs. IMiDs are a class of drugs that are used to treat multiple myeloma.The first chapter is an introduction to the clinical use of IMiDs, as well as the proteincereblon (CRBN), the primary target of IMiDs. The second chapter describes worktowards the identification of a novel IMiD target, WIZ, that is regulated by CRBNin an IMiD dependent manner. Mass spectrometry was performed to identify novelbinding partners, and IMiD dependent regulation by CRBN was validated usingchemical and genetic methods. Understanding how these drugs work will informthe production of more potent and more selective drugs.
", "doi": "10.7907/Z7PK-NH81", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11540", "collection": "thesis", "collection_id": "11540", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242019-115311540", "primary_object_url": { "basename": "Ferdinand_Huber_PhD_Thesis.pdf", "content": "final", "filesize": 112541653, "license": "other", "mime_type": "application/pdf", "url": "/11540/1/Ferdinand_Huber_PhD_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular Basis for Ribosomal Protein Protection from Cellular Degradation", "author": [ { "family_name": "Huber", "given_name": "Ferdinand Michael", "clpid": "Huber-Ferdinand-Michael" } ], "thesis_advisor": [ { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ribosomes are large macromolecular machineries composed of both protein and RNA constituents with a species-dependent molecular mass of at least ~3.3\u00a0MDa for the fully assembled eukaryotic 80S ribosome. Their catalytic activity is dependent on ribosomal RNA; therefore, ribosomes are bona fide ribozymes, and as such they mediate the final step of gene expression from DNA to RNA to protein by peptide bond formation between amino acids. Importantly, spatial separation of ribosome function and biogenesis into distinct cellular compartments allows for intricate regulatory mechanisms and rigorous quality control. Ribosome biogenesis occurs predominantly in the nucleolus and nucleus of the cell with final cytoplasmic maturation and quality control steps. Briefly, nucleolar ribosomal RNA together with ~200 trans-acting assembly factors co-transcriptionally forms the 40S and 60S pre-ribosomal subunits into which ~80 ribosomal proteins are incorporated in a hierarchical fashion.
\r\n\t\r\nRecent studies, including this thesis, have identified a novel class of dedicated ribosome assembly chaperones, in addition to the ~200 trans-acting ribosome assembly factors, which facilitate ribosomal protein shuttling. Ribosomal proteins are generated in the cytoplasm, and with only few exceptions they all have to enter the nucleus for incorporation into the pre-ribosomal subunits. Assembly chaperones can bind and protect unassembled ribosomal proteins either co-translationally or following nuclear import and shuttle them in a timely fashion to their destination sites at the maturing pre-ribosomal subunits. The first chapter of this thesis describes the identification and characterization of a dedicated assembly chaperone for the large ribosomal subunit protein RpL4, termed Acl4. Interestingly, Acl4 and likely also other dedicated assembly chaperones not only interact with ribosomal proteins to avoid aggregation and to shield them from unfavorable interactions, but also protect their client proteins from cellular degradation by the ubiquitin-proteasome machinery.
\r\n\t\r\nRibosomes are built by assembling equimolar amounts of ribosomal proteins, which generates a challenge for the cell to ensure stoichiometric quantities of ribosomal proteins. Recent studies have demonstrated that stoichiometric levels of ribosomal proteins are established by cellular degradation of excess protein via ubiquitination of unassembled components. The second chapter of this thesis describes a conserved degradation pathway, which is dependent on the E3 ubiquitin ligase Tom1 to mark unprotected and unassembled ribosomal proteins and target them for degradation. Moreover, it is demonstrated in the third chapter for the first time how an assembly chaperone protects its client ribosomal protein from ubiquitination and proteasome-mediated degradation. High resolution structures of the Acl4\u2022RpL4 complex as well as RpL4 in complex with the nuclear transport factor Kap104 visualize the molecular interactions of those proteins and uncover the molecular mechanism of protecting conserved Tom1-target sites within RpL4. Together, the reported results identify and characterize both a novel degradation pathway as well as a protection mechanism for ribosomal proteins and advance the understanding of the intricate regulation of ribosome biogenesis.
", "doi": "10.7907/4S7C-V170", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11413", "collection": "thesis", "collection_id": "11413", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02272019-142326751", "type": "thesis", "title": "Structure and Dynamics of HIV-1 Env Trimer", "author": [ { "family_name": "Wang", "given_name": "Haoqing", "orcid": "0000-0003-0277-3018", "clpid": "Wang-Haoqing" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The human immunodeficiency virus-1 (HIV-1) infects CD4+ helper T cells by fusing its lipid membrane to the host cell plasma membrane. HIV-1 Envelope (Env) glycoprotein, a trimer of gp120-gp41 heterodimers, is the fusion machinery that mediates viral and host cell membrane fusion, which is initiated by interaction between the host cell receptor CD4 and viral glycoprotein gp120. Receptor binding induces conformational changes in the Env trimer such that coreceptor CCR5/CXCR4 can bind to gp120 and trigger subsequent membrane fusion steps. Previous cryo-electron tomography (cryoET) and spectroscopy studies have shown that CD4 binding induces changes in Env trimer from the closed, prefusion conformation to an open, CD4-bound conformation. However, the CD4-bound Env trimer is dynamic and flexible such that structural biology study of CD4-bound Env trimer is difficult.
\r\n\r\nAs the sole glycoprotein located on HIV-1 viral surface, Env trimer is the only target for neutralizing antibodies. Since HIV-1 mutates rapidly, most neutralizing antibodies are strain-specific. However, a small percent of HIV-1 infected patients can develop broadly neutralizing antibodies (bNAbs) that neutralize a wide spectrum of viruses. In 2011, our collaborator isolated a bNAb called 8ANC195. To understand how 8ANC195 recognizes Env trimer and prevents infection, we used a combination of structural biology and biochemistry tools. Interestingly, we found that 8ANC195 can recognize Env trimer in both its prefusion and CD4-bound conformations by targeting an epitope at gp120-gp41 interface. Furthermore, 8ANC195 stabilizes the Env-CD4 complex, allowing us to investigate the conformational changes induced by CD4 in atomic resolution. By solving and comparing single-particle cryo-EM structures of Env trimer in complex with CD4 and/or antibodies, we found that: 1) CD4 binding induces coreceptor binding site exposure through a \u03b2-sheet rearrangements in each gp120 monomer. Several key residues allosterically regulate this coreceptor binding site exposure. 2) CD4 binding induces Env trimer opening together with gp41 conformational changes, which represents an intermediate between prefusion gp41 and postfusion gp41. 3) Env trimer opening is necessary but not sufficient for coreceptor binding site exposure, while CD4 binding induces both, some antibodies can open Env trimer without exposing the coreceptor binding site. These conclusions further illuminate how Env trimer mediates membrane fusion and inform potential strategies blocking viral entry.
\r\n", "doi": "10.7907/65VD-VM42", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11703", "collection": "thesis", "collection_id": "11703", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-035956740", "primary_object_url": { "basename": "WenChen_2019_Caltech-thesis.pdf", "content": "final", "filesize": 23199748, "license": "other", "mime_type": "application/pdf", "url": "/11703/1/WenChen_2019_Caltech-thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Proteomics Profiling and Functional Characterization of Caenorhabditis elegans Excreted/Secreted Proteins", "author": [ { "family_name": "Chen", "given_name": "Wen", "orcid": "0000-0001-8056-5711", "clpid": "Chen-Wen-Biochemistry-Molecular-Biophysics" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Excretory-secretory products (ESPs) are first characterized and defined in parasitic nematode proteomics studies as the combination of various biomolecules that are continuously excreted or secreted into the environment throughout the whole life cycle. ESPs are particularly interesting to many scientists as anti-parasitic vaccine candidates and\r\nas promising drug targets since large portions of ESPs are active enzymes that potentially function directly at the parasite-host or worm-environment interfaces. However, majority of the parasites lack whole genome sequence knowledge and genome-editing tools. Thus, the number of ESPs identified is limited and many functions of ES proteins cannot be elucidated. Therefore, we use the most studied nematode, Caenorhabditis elegans, as the model to characterize the composition of excreted/secreted proteins with the help of nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). We characterized more than 509 excreted/secreted proteins with mix-staged worms, including many metalloproteases, cysteine proteases, and lysozymes. Proteases and proteases inhibitors are a major group in C. elegans ESPs. We performed stable isotope dimethylvlabeling quantitative proteomics and compared C. elegans ESPs on different bacteria diets. Lysozymes are not only enriched in C. elegans ESPs but are also up-regulated in response to pathogen and bacteria.
\r\n\r\nComparative studies of expression profiles of developmental life stages and pathogen infections elucidate the dynamics in regulating ESP components. We successfully identified stage-specific ESP groups associated with L1, L3, adult, L2 dauer, and postdauer. We demonstrated that proteases activities are down regulated by increased protease inhibitor expressions, while during dauer exit proteases expressions are increased. The comparison between dauer excretome/secretome and RNA-seq dauer expression profiles revealed 91 ESP encoding genes that are highly expressed in dauers. We performed dauer formation assay to these dauer-associated gene mutants. The great prediction rate confirmed that our comparative method is the simplest way to quickly pick out candidates for functional assays. Similarly, we employed this comparative method to pathogeninduced transcriptomes. We reported a group of genes that are associated with Serratia marcescens infection and a group of bacterial pathogens responding genes. We confirmed the roles of C. elegans ESPs in immuoregulation by infection assays with various pathogens. Lysosomes and cysteine protease inhibitor are among the most important genes in innate immune response pathway of C. elegans defending pathogen infection.
\r\n\r\nThe recent discovery of a C. elegans sibling species, Caenorhabditis inopinata, allows the deeply comparative study for evolutional interpretation. The excretome/secretome of C. inopinata has not been characterized. We took advantage of the sensitive and highthroughput technique of nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) to directly characterize the protein components of C. inopinata excretome/secretome. Functional annotations reveal several protein families, including C-type lectins, Cathepsin Z, Cathepsin B family, transthyretin, and saposin-like families, suggesting ESPs play critical roles in regulating innate immune response. We compared C. inopinata excretome/secretome with C. elegans. The structures are highly conserved across species, suggesting the sibling species share common mechanism to respond to environmental stimuli.
", "doi": "10.7907/WTMK-7M75", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11713", "collection": "thesis", "collection_id": "11713", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-131615199", "primary_object_url": { "basename": "mosesso_richard_2019_thesis_proofread.pdf", "content": "final", "filesize": 9464813, "license": "other", "mime_type": "application/pdf", "url": "/11713/1/mosesso_richard_2019_thesis_proofread.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanistic Investigations of Receptor Signaling via Canonical and Non-Canonical Amino Acid Mutagenesis", "author": [ { "family_name": "Mosesso", "given_name": "Richard Albert", "orcid": "0000-0003-0927-0843", "clpid": "Mosesso-Richard-Albert" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes investigations of the mechanisms by which pentameric ligand-gated ion channels (pLGICs) activate (\"gating\") using canonical and non-canonical amino acid mutagenesis. Chapter 1 provides an introduction to the systems being studied, their physiological roles, and the techniques that we have used to study them. Chapter 2 describes a series of experiments comparing the roles of amino acid residues proximal to the neurotransmitter binding site in the type 3 serotonin receptor (5-HT3R) to the aligning residues of the muscle-type nicotinic acetylcholine receptor (nAChR). The findings presented in Chapter 3 assess the functional roles of proline residues in the prokaryotic pLGIC, Erwinia ligand-gated ion channel (ELIC). Chapter 4 describes an extensive investigation of salient details of 5-HT3R gating using canonical and non-canonical amino acid mutagenesis of amino acid residues at the interface of the extracellular domain and transmembrane domain of this receptor. Chapter 5 applies a photocrosslinking strategy employing the non-canonical amino acid p-azidophenylalanine to study dimerization and cofactor interactions of the estrogen receptor \u03b1.
", "doi": "10.7907/X18Z-XE16", "publication_date": "2019-06-14", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11716", "collection": "thesis", "collection_id": "11716", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-145818414", "primary_object_url": { "basename": "AEM_Blom_2019_0530_Full_Thesis.pdf", "content": "final", "filesize": 7524685, "license": "other", "mime_type": "application/pdf", "url": "/11716/1/AEM_Blom_2019_0530_Full_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Functional Evaluation and Development of Novel Agonists and Modulators of Neuronal Ion Channels", "author": [ { "family_name": "Blom", "given_name": "Antoinette Elisabeth Maria", "orcid": "0000-0002-7441-4893", "clpid": "Blom-Antoinette-Elisabeth-Maria" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "clpid": "Shapiro-M-G" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation describes studies of activation of neuronal ion channels and evaluating new ligands to modulate this process. In chapter two, we expanded the binding model of cytisine to the \u03b14\u03b22 nicotinic acetylcholine receptor. We also determined how C(10)-modification of cytisine impacts the key binding interactions between cytisine and its binding site. To achieve this, we used non-canonical amino acid mutagenesis to probe the electrostatic binding interactions of a novel series of C(10)-cytisine derivatives. In order to perform similar studies in the \u03b13\u03b24 nAChR subtype, we describe the heterologous expression of mouse and human \u03b13\u03b24 nAChRs in Xenopus Laevis oocytes in appendix one. Chapter three describes the development and functional evaluation of a novel series of pyrrolidinoindolines for agonism and modulation of the GABAA receptor. Additionally, we performed mutagenesis studies to identify the binding site of these novel ligands. Appendix two describes a different screen for activation or modulation of GABAA receptors using a set of phenolic compounds implicated in Autism Spectrum Disorder. Chapter four shifts focus to voltage-gated ion channels: in this chapter, the ultimate goal was to photochemically control the activation of VGSCs and make progress towards developing a RubpyC17-based photoswitch that could be used in an artificial retina. To this end, we determined the functional effects of several ruthenium bipyridine analogs on voltage-gated sodium and potassium channels.
", "doi": "10.7907/RG56-G044", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11718", "collection": "thesis", "collection_id": "11718", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072019-153337463", "primary_object_url": { "basename": "Thesis-Yandong Zhang-Final.pdf", "content": "final", "filesize": 2182617, "license": "other", "mime_type": "application/pdf", "url": "/11718/1/Thesis-Yandong Zhang-Final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Highly Multiplexed Imaging of E. Coli Chromosome and Sensitive Detection of Single-Cell Protein", "author": [ { "family_name": "Zhang", "given_name": "Yandong", "orcid": "0000-0003-3291-9209", "clpid": "Zhang-Yandong" } ], "thesis_advisor": [ { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The driving force for biology research is the development of new techniques which allow high-sensitivity, high-throughput measurement in various contexts. Over the past decade, the emerging of a variety of single-cell techniques have greatly transformed our understanding of biological system. My thesis work was therefore focused on development of new single- cell techniques and use the techniques to generate new insights into biological system. Specifically, in the first part of my thesis work, we developed DNA seqFISH, a technique that allows us to image more than 100 different loci on the chromosome in single cells. We applied this technique to image E. coli chromosome with 50kb genomic resolution and 50nm spatial precision. Our data allows us to parse the E. coli chromosome structure according to their different spatial conformations and different cell-cycle stages. We identified two chromosome conformations with distinct domain structures, which is obscured from previous population-average research. We further characterized the domain structure dynamics during daughter chromosome segregation. Therefore, our data provides a high- resolution, dynamic view of E. coli chromosome structure.
\r\n\r\nIn the second part, we developed a novel method for sensitive detection of targeted protein and its post-translational modification (PTM) isoform in single cells. Instead of depending on antibodies to distinguish targeted protein and its PTM isoform, we developed an efficient covalent barcoding strategy to barcode targeted protein inside the cells. Thereafter, targeted protein and its PTM isoform are separated by conventional gel electrophoresis, while their single-cell identity is preserved in the covalently attached oligo. By counting the attached DNA oligos using next-generation sequencing, targeted protein, and its PTM isoform can be accurately measured. We demonstrated the utility of the technology by quantification of histone protein, H2B and its mono-ubiquitination isoform, H2Bub at single-cell level. Our method revealed the single-cell heterogeneities of H2Bub/H2B ratio and its cell-cycle dynamics. Our method therefore provides an antibody-free method for sensitive detection of proteins and its isoforms in single cells.
", "doi": "10.7907/CDSX-MR28", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11561", "collection": "thesis", "collection_id": "11561", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292019-142906583", "primary_object_url": { "basename": "Thesis_Jae-Ho-Lee_Final.pdf", "content": "final", "filesize": 23848880, "license": "other", "mime_type": "application/pdf", "url": "/11561/1/Thesis_Jae-Ho-Lee_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanisms of Co-Translational Protein Targeting by Mammalian SRP", "author": [ { "family_name": "Lee", "given_name": "Jae Ho", "orcid": "0000-0002-8663-3209", "clpid": "Lee-Jae-Ho" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Hoelz", "given_name": "Andre", "clpid": "Hoelz-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Proper biogenesis of nascent protein is essential for cell survival. Signal recognition particle (SRP) is an essential and universally conserved factor involved in biogenesis of ~30% of the proteome through co-translational targeting of nascent proteins to Endoplasmic Reticulum (ER). Despite its importance, the mechanisms by which eukaryotic SRP ensure selective and efficient delivery of substrates to ER is poorly understood. Here, we reconstituted human SRP and SRP receptor (SR) to study the interaction between human SRP and SR, and conformational dynamics of SRP and SRP-SR targeting complex through biochemical and biophysical methods. We find that signal sequence and ribosomal components of the substrate sequentially activate human SRP. Especially, presence of signal sequence pre-organizes SRP conformation for efficient recruitment of SR, allowing specific and efficient targeting. In addition, we discover two essential roles of a conformational change in SR, where it is required not only for brining targeting complex near the ER membrane, but also for inducing subsequent conformational changes of SRP-SR complex that ensures proper delivery of substrates to Sec61 translocon on ER membrane.
", "doi": "10.7907/RF8Y-DK59", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11071", "collection": "thesis", "collection_id": "11071", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112018-200314076", "primary_object_url": { "basename": "EOB Thesis 062018c.pdf", "content": "final", "filesize": 11150005, "license": "other", "mime_type": "application/pdf", "url": "/11071/1/EOB Thesis 062018c.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Redox Signaling in Eukaryotic DNA Replication and Repair", "author": [ { "family_name": "O'Brien", "given_name": "Elizabeth", "orcid": "0000-0003-2889-1688", "clpid": "O'Brien-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_chem" } ], "abstract": "DNA-mediated charge transport chemistry (DNA CT) offers an intriguing regulatory mechanism in biology, as it is long-range, rapid, and sensitive to mismatches and perturbations to base stacking. DNA-processing enzymes in all three domains of life moreover have been shown to contain [4Fe4S] clusters, commonly redox cofactors. Bacterial [4Fe4S] repair proteins have been shown to signal one another using long-range DNA-mediated charge transport (DNA CT), facilitating the redistribution to damaged genomic DNA in cells. The role of metabolically expensive, [4Fe4S] cluster cofactors in eukaryotic systems, however, was less clear than in prokaryotes.
\r\n\r\nHere we examine the chemical role of the [4Fe4S] cluster in eukaryotic DNA primase and the human base excision repair glycosylase, MUTYH. The primase cluster functions as a redox switch regulating DNA binding and redox signaling activity in humans and yeast. Yeast moreover require the primase redox switch for viability. Human MUTYH, a bifunctional glycosylase which repairs oxidative DNA lesions, performs DNA-mediated redox signaling, similarly to the bacterial homologue MutY. The MUTYH mutation which destabilizes the [4Fe4S] cluster during redox signaling, C306W, promotes degradation and loss of activity, associated with hereditary colorectal cancer.
\r\n\r\nTo assess the redox role of the human primase [4Fe4S] cluster, we perform anaerobic DNA electrochemistry on the [4Fe4S] domain of human primase (p58C), which independently binds DNA. On DNA-modified Au electrodes, we compare the redox activity of electrochemically oxidized and electrochemically reduced p58C. Oxidized [4Fe4S]3+ p58C is electrochemically active, and reduced [4Fe4S]2+ p58C state is redox-inert. This redox-driven switch is electrochemically reversible, and is mediated by a triad of conserved tyrosines between the DNA binding interface and [4Fe4S] cluster. Mutation of residues Y309, Y345, and Y347 to phenylalanine causes attenuation of redox switching on DNA. Single-atom mutations in the redox pathway moreover compromise initiation and truncation of primer synthesis but do not affect RNA polymerase activity. We find that primase truncation is gated by DNA CT in vitro; a single mismatch in the nascent primer abrogates truncation of primase products. As\r\nprimase is tethered to DNA polymerase \u03b1, a putative [4Fe4S] enzyme to which primase hands off the RNA-primed template, we propose that DNA-mediated signaling between primase and polymerase \u03b1 chemically regulates this handoff during the first steps of replication.
\r\n\r\nEukaryotic primase must bind both DNA and nucleotide triphosphates (NTPs) in order to convert to active form. Using DNA electrochemistry we show that p58C, and full-length DNA primase, display a robust, semi-reversible NTP-dependent signal on DNA, centered near 150mV vs. NHE. This signal is dependent on the tyrosine redox pathway. The presence of reversible redox activity at a physiological potential when primase is bound to DNA and NTPs suggests that reversible redox switching from the [4Fe4S]2+ to the [4Fe4S]3+ state is important for the activity of primase during replication.
\r\n\r\nThe cluster serves as a redox switch governing DNA binding in yeast primase, just as in human primase. Mutation of tyrosines 395 and 397 in yeast primase moreover, alters the same electron transfer chemistry as the mutation of their orthologues, Y345 and Y347, respectively, alters in human primase. Although these tyrosines are arranged differently in the yeast and human proteins, they perform the same reaction to affect the switch. The single-atom Y395F mutation causes some sensitivity to chemically induced oxidative stress in yeast, and single-residue mutation Y397L confers lethality in yeast cells. A constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases, regulates the affinity for RNA-primed DNA template, and is required for primase function in vivo.
\r\n\r\nWe finally characterize a novel mutation in the [4Fe4S] human base excision repair protein, MUTYH, which destabilizes the cluster environment and has pathogenic consequences. The MUTYH C306W mutation alters one of the cysteines coordinating the cluster to tryptophan. This mutation moreover is associated with hereditary colorectal cancer and causes defective DNA binding and enzymatic activity. We perform DNA electrochemistry on WT MUTYH, as well as C306W and two cancer-associated mutants, Y197C and G396D, which have an unaltered cluster environment. MUTYH variants participate in redox signaling, but C306W is destabilized upon oxidation from the [4Fe4S]2+ to the [4Fe4S]3+ state during signaling on DNA, leading to degradation to a [3Fe4S]+ cluster and loss of DNA binding and activity. A [4Fe4S] human DNA repair enzyme performs redox signaling on DNA; dysregulation of this signaling activity is linked to tumorigenesis.
\r\n", "doi": "10.7907/KGCP-SD98", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10443", "collection": "thesis", "collection_id": "10443", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09202017-130540216", "primary_object_url": { "basename": "Buscagan_Trixia_2018_Thesis.pdf", "content": "final", "filesize": 8885097, "license": "other", "mime_type": "application/pdf", "url": "/10443/1/Buscagan_Trixia_2018_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Transition Metals as Catalysts for Cross-Coupling and Dinitrogen Fixation", "author": [ { "family_name": "Buscagan", "given_name": "Trixia Marie", "orcid": "0000-0001-8242-9203", "clpid": "Buscagan-Trixia-Marie" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Fu", "given_name": "Gregory C.", "clpid": "Fu-G-C" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transition metals are used as catalysts in the laboratory and by nature to facilitate difficult chemical transformations. Herein, three different metal containing catalysts are discussed: Pd and Ni catalysts towards the formation of carbon-carbon (C-C) bonds and Fe catalysts towards the reduction of N2 to NH3.
\r\n\r\nIn Chapter 2, mechanistic studies of Pd- and Ni-catalyzed cross-coupling reactions are discussed. The mechanism of transmetalation of a Pd-catalyzed Suzuki cross-coupling reaction is studied using a stereochemical probe, revealing that transmetalation occurs with retention of configuration, consistent with transmetalation occurring through a frontside-attack mechanism. Next, to explore the viability of a transmetalation first pathway in an asymmetric Negishi cross-coupling reaction, S = 1/2 NiIBr and NiI\u2013alkyl complexes were synthesized, crystallographically characterized, and their reactivities explored. Based on these reactivity studies, evidence against a transmetalation first pathway is provided using a variety of spectroscopic methods.
\r\n\r\nIn Chapter 3, new Fe(N2)(H)x complexes are synthesized. These complexes catalyze the reduction of N2 to NH3 and the yields for NH3 are improved if the reactions are performed in the presence of Hg lamp photolysis. Preliminary mechanistic studies exploring the role of light are discussed. In the final chapter, new ligand scaffolds are developed that can bind a Lewis acidic and Lewis basic metal center. These ligand frameworks support one- and two-atom bridges between the two metal sites. Finally, we discovered that some of the new complexes are catalysts for N2 to NH3 reduction and olefin hydrogenation.
", "doi": "10.7907/Z98P5XPM", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10353", "collection": "thesis", "collection_id": "10353", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07282017-132944334", "primary_object_url": { "basename": "PDF (Akagi Thesis - Full).pdf", "content": "final", "filesize": 6377119, "license": "other", "mime_type": "application/pdf", "url": "/10353/43/PDF (Akagi Thesis - Full).pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Biosynthesis of Ascarosides in Caenorhabditis elegans", "author": [ { "family_name": "Akagi", "given_name": "Allison Emi", "orcid": "0000-0002-7233-6190", "clpid": "Akagi-Allison-Emi" } ], "thesis_advisor": [ { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ascarosides comprise a family of small signaling molecules that have been shown to regulate important events and behaviors in the life history of the nematode Caenorhabditis elegans. Although the different roles of individual ascarosides appear to be determined by the variances in chemical structure, the mechanisms by which ascarosides are synthesized as well as the locations in which ascarosides are produced within the worm are largely unknown. In this thesis, we examined ascaroside production in the intestine, hypodermis, and body wall muscle of the worm by driving the expression of the protein DAF-22 under different tissue-specific gene promoters. While the body wall muscle and hypodermis are capable of synthesizing ascarosides, the intestine appears to be the major site of pheromone production. Additionally, we found through transgenic rescue and HPLC-MS analysis, that the acyl-CoA synthetase ACS-7 plays a significant role in the addition of moieties derived from primary metabolic pathways to the 4\u2019-position of the ascarylose sugar core of ascr#9.
", "doi": "10.7907/Z9ZK5DVT", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10354", "collection": "thesis", "collection_id": "10354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07282017-141924517", "primary_object_url": { "basename": "TJZ Thesis Final Proofread and Corrected.pdf", "content": "final", "filesize": 7024906, "license": "other", "mime_type": "application/pdf", "url": "/10354/23/TJZ Thesis Final Proofread and Corrected.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Magnetic Field Effects and Biophysical Studies on DNA Charge Transport and Repair", "author": [ { "family_name": "Zwang", "given_name": "Theodore Joseph", "clpid": "Zwang-Theodore-Joseph" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA-mediated charge transport (DNA CT) is well established in both ground and excited state systems. Although theoretical models are still being developed, it is clear that the integrity of the extended \u03c0-stack of the aromatic heterocycles, the nucleic acid bases, plays a critical role. Electron donors and acceptors must be electronically well coupled into the \u03c0-stack, typically via intercalation. Perturbations that distort the \u03c0-stack, such as single-base mismatches, abasic sites, base lesions, and protein binding that kinks the double helix, attenuate DNA CT dramatically.
\r\n\r\nThis thesis encompasses work that first aims to understand how DNA duplex structure informs characteristics of DNA CT and then continues to develop an understanding of the role these structural features play in biological systems. To contextualize these advancements, this first chapter outlines foundational work that has shown ways that DNA structure influences its ability to conduct charge.
\r\n\r\nNext, experiments were conducted on magnetized DNA-modified electrodes to explore spin-selective electron transport through hydrated duplex DNA. These results show that the two spins migrate through duplex DNA with a different yield and that spin selectivity requires charge transport through the DNA duplex. Significantly, shifting the same duplex DNA between right-handed B- and left-handed Z-forms leads to a diode-like switch in spin selectivity; which spin moves more efficiently through the duplex depends upon the DNA helicity. With DNA, the supramolecular organization of chiral moieties, rather than the chirality of the individual monomers, determines the selectivity in spin, and thus a conformational change can switch the spin selectivity.
\r\n\r\nThis exquisite spin selectivity begged the question: how might biology take advantage of such a spin filter? Photolyase and cryptochromes both have been shown to exhibit magnetosensitive chemistry nearby a DNA binding pocket, and photolyase had previously been shown capable of DNA CT. Thus, electrochemical studies were conducted to monitor the repair of cyclobutane pyrimidine dimer lesions by E coli photolyase and truncated A Thaliana Cryptochrome 1 with an applied magnetic field. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 Gauss, though spin selective DNA CT is not involved. These data illustrate how cyclobutane dimer repair could be used in a biological compass that is informed by the angles of Earth\u2019s magnetic field.
\r\n\r\nNext DNA-mediated electrochemistry and atomic force microscopy studies were used to describe a role for redox active [4Fe4S] clusters in DNA-mediated charge transport signaling. DNA-modified electrochemistry shows that the [4Fe4S] cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III, a base excision repair enzyme, cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. These data are then described using an equilibrium model which elucidates fundamental characteristics of this redox chemistry that allow DNA CT to coordinate the activities of DNA repair enzymes across the genome.
\r\n\r\nThe importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process is then further explored. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.
", "doi": "10.7907/Z9TT4P4H", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10840", "collection": "thesis", "collection_id": "10840", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04302018-143201092", "primary_object_url": { "basename": "180430_final_thesis.pdf", "content": "final", "filesize": 3655146, "license": "other", "mime_type": "application/pdf", "url": "/10840/1/180430_final_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structural and Functional Characterization of the Escherichia coli MetNI Methionine Transporter\r ", "author": [ { "family_name": "Nguyen", "given_name": "Phong Thanh", "orcid": "0000-0002-6390-7350", "clpid": "Nguyen-Phong-Thanh" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer partner in the inward facing conformation (Kadaba et al. Science 321, 250-253, 2008), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP and EDTA to stabilize MetNI in the ATP-bound conformation, and the N229A variant of the binding protein MetQ to disrupt methionine binding as shown in this work, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. We then solved a 2.95 \u00c5 resolution crystal structure of the outward-facing conformation of the MetNI transporter, in complex with its binding protein, MetQ. The structure sheds light on how the C-regulatory domains regulate transport activity by rearrangement of a hydrogen bonding network between their interfaces in two different conformations. Structure of the substrate-free homologous MetQ from N. meningitides was also resolved using the N-to-A mutation (N238A). Superimposition of the substrate-bound, substrate-free (homologous model) MetQ and the binding protein MetQ in complex with its MetNI transporter (complexed MetQ) reveals unexpected structural features of the complexed MetQ, indicates a different substrate delivery mechanism for the MetNI transporter. These structural insights, coupled with thermodynamic binding constant and in vivo transport studies, support an unconventional transport mechanism for the Type-I methionine ABC importer.\r\n", "doi": "10.7907/x9ed-9n64", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10892", "collection": "thesis", "collection_id": "10892", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152018-143529378", "type": "thesis", "title": "Harnessing Biological Tools of Protein Transport and Catalysis", "author": [ { "family_name": "McAvoy", "given_name": "Camille Zandra", "orcid": "0000-0002-9828-1538", "clpid": "McAvoy-Camille-Zandra" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This work covers two projects related to protein structure and function. The first focuses on studies of chloroplast signal recognition particle 43 (cpSRP43), its interaction with substrate (the light-harvesting, chlorophyll-binding proteins, or LHCP), the role of conformational change in its activity, and the use of cpSRP43 as a tool for handling nonnative proteins. This work utilizes a variety of biochemical and biophysical approaches including light scattering and electron paramagnetic resonance to probe the structure-function relationship of cpSRP43. The second project entails the study of the C-C bond formation mechanism of nitrogenase, a biological nitrogen fixer found in soil microorganisms. Together these projects make for an interesting story of the medicinal and agricultural applications of basic biochemistry.
", "doi": "10.7907/EXJK-C379", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10941", "collection": "thesis", "collection_id": "10941", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252018-140505343", "primary_object_url": { "basename": "Arias_thesis_29May18.pdf", "content": "final", "filesize": 4185649, "license": "other", "mime_type": "application/pdf", "url": "/10941/9/Arias_thesis_29May18.pdf", "version": "v14.0.0" }, "type": "thesis", "title": "Examination of Selenium Incorporation and Product Formation in the Nitrogenase FeMo-Cofactor", "author": [ { "family_name": "Arias", "given_name": "Renee Justine", "orcid": "0000-0002-6505-7513", "clpid": "Arias-Renee-Justine" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nitrogenase is the only known enzyme to convert the triply bonded atmospheric dinitrogen (N2) to bioavailable ammonia (NH3) in an ambient environment, breaking one of the strongest chemical bond in nature in the process. Industrially, the Haber-Bosch process is also capable of reducing dinitrogen to ammonia, and is essential for worldwide food production 1,2. Due to the high temperatures and pressures required for the Haber-Bosch process (between 300-550\u00baC and 15-25 MPa) and its requirement for molecular hydrogen, it has become paramount to scientifically investigate the biological processes of nitrogen fixation to ultimately develop more efficient methods to produce bioavailable ammonia. Nitrogenase utilizes two component proteins, the Fe-protein and the MoFe-protein, to reduce ammonia in an ATP-hydrolysis dependent and electron-intensive reaction. Besides the canonical dinitrogen reduction reaction, nitrogenase can reduce a variety of other substrates including: acetylene (C2H2), carbon dioxide (CO2), carbon monoxide (CO), carbonyl sulfide (COS), nitrous oxide (N2O), diazene (N2H2), and more 3-11. CO has long been of interest to the study of the mechanism of nitrogenase, owing to its isoelectronic identity to N2, and its potent inhibitor properties at well as its ability to serve as a weak substrate 12,13. Like CO, cyanide compounds (X-CN) are also of interest to the study of nitrogenase due to the isoelectronic nature of CN- to N2. However, cyanide compounds serve as particularly interesting spectroscopic and crystallographic tools, because X in X-CN can be substituted for more significant sulfur or selenium (Se). In this study, we investigate the substrate properties of SeCN-, with Se-incorporation into the active site FeMo-cofactor and concurrent reduction of SeCN- to methane (CH4). This study serves as yet another link between substrate reduction in nitrogenase. Part of this work describes the incorporation of Se into the cofactor as a vehicle for high-resolution study of nitrogenase under turnover using spectroscopy and crystallography, while another part describes a proposal for future work on the trapping of enzyme intermediates by fast-growing crystallography.
", "doi": "10.7907/5WZV-R440", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:11069", "collection": "thesis", "collection_id": "11069", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112018-181603942", "type": "thesis", "title": "Interrogating the Structural Landscape of Malaria Biomarkers with Epitope Targeted Peptide Capture Agents", "author": [ { "family_name": "Liang", "given_name": "JingXin", "orcid": "0000-0001-6600-8409", "clpid": "Liang-JingXin" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Antibodies have conventionally been used as molecular recognition agents against epitopes, or antigenic regions, for protein capture and detection. The ability of monoclonal and polyclonal antibodies to selectively bind their targets with high affinities makes them excellent agents for specific protein recognition. However, as large proteins themselves (~150 kDa), antibodies are susceptible to changes in pH, temperature, and biochemical environment, particularly proteolytic cleavage. Additionally, epitope binding on antibodies is reliant on their rigid tertiary structure to position key functional groups that facilitation antigen recognition. Retaining the integrity of the protein structure creates rigid limitations against chemical modifications of antibodies to suit unique needs.
\r\n\r\nProtein-catalyzed capture agents (PCCs) developed within the Heath group at Caltech address the limitation of antibodies as affinity agents. Using epitope-targeted in situ click screening methodology, the Heath group has developed peptidomimetic molecules that offer an alternative solution to antibodies. These PCCs exhibit high affinity and selectivity for their protein targets. As peptide-based molecules, PCCs can be engineered to be biochemically stable and resistant to changes in their chemical environment. Their peptide-based structures are readily amenable to chemical modifications and allow for adaptation to a range of applications.
\r\n\r\nThis thesis describes the development of PCCs against unique protein biomarkers for the detection of the most lethal species of malaria infection, Plasmodium falciparum. Malaria is a global health epidemic and its eradication is reliant on rapid and accurate diagnostics for prompt treatment. We targeted the P. falciparum specific biomarkers lactate dehydrogenase (LDH) and Histidine-rich protein 2 (HRP2), both of which present unique challenges for protein capture. The LDH biomarker is homologous across malaria species, whereas HRP2 is highly polymorphic and lacks distinct secondary structure. The variation in sensitivity of HRP2 detection by antibody-based tests has been attributed to the genetic polymorphism of the biomarker.
\r\n\r\nIn Chapter 1, we describe the development of high affinity PCCs that bind selectively to the LDH biomarker. We targeted an epitope that was highly homologous across LDH species. This chapter also details the expansion of mono-valent PCC agents into bivalent ligands using the protein architecture to select secondary ligands for binding improvement. For the HRP2 biomarker, we developed a multiple epitope targeting strategy to address protein polymorphism. We targeted for epitopes in HRP2 and developed PCCs that bind in the range of monoclonal antibodies.
\r\n\r\nChapter 2 details the expansion of PCC agents developed against HRP2 into multivalent molecules for improved binding. The development of bivalent ligands from combinatorial screening of linker libraries is presented. The optimal linker lengths determined by the screens are described.
\r\n\r\nIn Chapter 3, a general strategy for targeting the protein landscape to inhibit formation of a protein and biomolecule complex with PCCs against HRP2 is demonstrated. Specifically, the inhibition of heme sequestration by HRP2 is shown. A bivalent ligand that targets two epitopes on HRP2 is shown to have enhanced inhibitory potency over any single or cocktail combination of PCCs.
\r\n\r\nAltogether, the studies herein demonstrate the utility of peptidomimetic molecules as agents for protein capture and detection as well as a generalizable strategy of functional inhibition through epitope-targeting.
", "doi": "10.7907/rxtr-6152", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10125", "collection": "thesis", "collection_id": "10125", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04022017-212739773", "type": "thesis", "title": "Biophysical Characterization of an ABC L-methionine Transporter", "author": [ { "family_name": "Li", "given_name": "Qi Wen", "orcid": "0000-0001-9493-2316", "clpid": "Li-Qi-Wen" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Goentoro", "given_name": "Lea A.", "clpid": "Goentoro-L-A" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ATP-binding cassette (ABC) superfamily is pivotal to a number of important biochemical processes and ubiquitous in all kingdoms of life. Previous studies of ABC transporters have been heavily focused on the structural determination of the different intermediates of the transport cycle. In order to characterize the mechanism of an E. coli L-methionine transporter, which is an ABC importer, we first collated previously reported structural information on the conformational states of several well characterized ABC importers and associated binding proteins, and identified four major conformations (i.e., pre-T, outward, post-T, and inward state). We stabilized these intermediates using appropriate mutations, substrates, and nucleotides. We then studied the kinetics and thermodynamics of the formation of these states using surface plasmon resonance (BiaCore, GE Healthcare) and MicroScale Thermophoresis (NanoTemper). We developed a quantitative model that details the kinetic and molecular mechanism of E. coli MetNI. Towards this goal, we extended the Two-State, alternating access model to include other intermediates that are crucial to transport and are using this to provide a temporal understanding of transport. While this model is developed to describe the behavior of the Lmethionine MetNI importer, it may also have predictive power for other ABC Type I importers, since the NBD\u2019s response for coupling transport to ATP-binding and hydrolysis are highly conserved in this family.
", "doi": "10.7907/Z9125QN9", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10253", "collection": "thesis", "collection_id": "10253", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022017-112043547", "primary_object_url": { "basename": "HunterBryan2017thesis.pdf", "content": "final", "filesize": 22940679, "license": "other", "mime_type": "application/pdf", "url": "/10253/73/HunterBryan2017thesis.pdf", "version": "v16.0.0" }, "type": "thesis", "title": "Fuels and Materials from Sunlight and Water", "author": [ { "family_name": "Hunter", "given_name": "Bryan Michael", "orcid": "0000-0001-8559-9304", "clpid": "Hunter-Bryan-Michael" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rossman", "given_name": "George Robert", "clpid": "Rossman-G-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "Resnick Sustainability Institute" }, { "literal": "div_chem" } ], "abstract": "The urgency to develop new technologies that harness energy and natural feedstocks in a sustainable fashion has never been more apparent. With global power consumption growing at an exponential rate, only one resource is truly capable of powering the planet: the sun. Sunlight is reliable, clean, and free.
\r\n\r\nSignificant resources have been pledged to develop and refine solar energy devices that convert photons into electricity (i.e. photovoltaics), but the sun\u2019s intermittency and the poor overlap of solar irradiance with global power demand a different strategy. In light of these limitations, we have proposed a device which converts solar energy into reduced chemical fuels (e.g. dihydrogen or methane) that can be indefinitely stored and easily transported. In principle, the only required inputs are sunlight, an earth-abundant feedstock such as carbon dioxide, protons (H+), and reducing equivalents (e-). The source of these protons and electrons must be abundant and ubiquitous\u2014we chose water.
\r\n\r\nDespite the 2-billion-year history of plants performing water oxidation to produce molecular oxygen, protons, and electrons (Photosystem II), our understanding of this complex 4H+/4e- process has been severely limited. Only recently have high-performing, earth-abundant heterogeneous electrocatalysts been reported that can be scaled up to make functioning devices.
\r\n\r\nThis dissertation describes progress on both the synthetic and mechanistic fronts in developing earth-abundant heterogeneous water oxidation catalysts for solar-driven water splitting. We have synthesized nanoparticulate Ni-Fe catalysts with the highest measured activity on flat electrodes to date. We carefully characterized these materials spectroscopically to determine that edge-site iron was active in catalysis. We then undertook novel in-situ spectroelectrochemical techniques in non-aqueous media to identify the active iron species, which is surprisingly a cis-dioxo-iron(VI) corner site. The data also indicate that geminal iron-oxo coupling may be the operative mechanism of O-O bond formation, a new scheme with potential biological relevance.
\r\n\r\nFinally, we have expanded our goal to include sustainably reducing other feedstocks, such as carbon dioxide and hydrocarbons. In doing so, we aim to make pharmaceuticals, polymers, and other high-value products from sunlight and water.
", "doi": "10.7907/Z9FQ9TNB", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10230", "collection": "thesis", "collection_id": "10230", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05312017-133325449", "primary_object_url": { "basename": "THESIS.pdf", "content": "final", "filesize": 4208773, "license": "other", "mime_type": "application/pdf", "url": "/10230/1/THESIS.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Physiological and Biochemical Mechanisms of Phenazine-Mediated Survival in Pseudomonas aeruginosa", "author": [ { "family_name": "Glasser", "given_name": "Nathaniel Robert", "orcid": "0000-0002-2833-5166", "clpid": "Glasser-Nathaniel-Robert" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The opportunistic pathogen Pseudomonas aeruginosa secretes a class of colorful redox-active small molecules known as phenazines. Numerous functions have been proposed for phenazines, including antibiotic activity, virulence, cell-to-cell signaling, iron acquisition, and survival. This thesis delves into mechanisms of the latter role, that of long-term survival under oxidant-limiting conditions. Using a diverse array of methods, I investigated how phenazines support survival and how cells transfer electrons to phenazines, as well as the downstream effects that phenazines have on P. aeruginosa.
\r\n\r\nDirect measurements of NAD(H), ATP, the membrane potential, and fermentation products revealed that phenazines promote redox homeostasis and subsequently ATP synthesis. The ATP is used to maintain a membrane potential through the reverse action of the ATP synthase complex. Even though P. aeruginosa does not ferment on sugars, phenazines enable the anaerobic oxidation of glucose to acetate, suggesting P. aeruginosa may have previously under-appreciated metabolic flexibility in the absence of terminal electron acceptors. Activity assays with proteins purified natively from P. aeruginosa showed that glucose oxidation might be enabled in vivo by the pyruvate dehydrogenase complex, which can directly reduce phenazines using pyruvate as an electron donor. Liquid chromatography and mass spectrometry of culture supernatants showed that phenazines alter the chain length distribution of secreted quinolones, which may have indirect downstream signaling effects. Based on this result, combined with data from survival experiments, I hypothesize that phenazine-mediated redox homeostasis promotes \u03b2-oxidation and that fatty acid metabolism contributes to long-term survival. Further analysis also showed that P. aeruginosa cultures contain several previously-unreported sulfonated phenazines. In its natural environment, P. aeruginosa undoubtedly encounters other microbial species that consume or modify its phenazines. At least one of these, a Mycobacterium, contains a pyocyanin demethylating enzyme. The X-ray crystal structure of this protein revealed a novel reaction mechanism wherein the substrate is its own electron acceptor. Together, this work illuminates some of the many ways phenazines shape microbial communities in both clinical and environmental contexts.
\r\n", "doi": "10.7907/Z9SN070S", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10335", "collection": "thesis", "collection_id": "10335", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06122017-230026717", "type": "thesis", "title": "DarwinDock and GAG-Dock: Methods and Applications for Small Molecule Docking", "author": [ { "family_name": "Griffith", "given_name": "Adam Reid", "clpid": "Griffith-Adam-Reid" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Computational modeling is an effective tool in studying complex biological systems. Docking of small molecule ligands in particular is useful both in understanding the functioning of proteins as well as in the development of pharmaceuticals. Together with experiment, modeling can often provide a thorough picture of a given system. Computation can often provide details that are difficult or impossible to determine experimentally, while experiments provide guidance on what calculations are useful or interesting. Our goal is to extend computational modeling, specifically ligand docking, to systems not previously possible, such as the challenging glycosaminoglycan (GAG) systems. In order to do this it was first necessary to develop an automatic way of performing docking without extensive user input and experimental knowledge to narrow the list of candidate poses. DarwinDock represents our efforts in this respect. It is a method for small-molecule docking that separates pose generation and scoring into separate stages, which allows for complete binding site sampling followed by efficient, hierarchical sampling. Our convergence criteria for complete sampling allows for diverse systems to be studied without prior knowledge of how large a set of poses needs to be to span a given binding site, making the procedure more automatic. We also replace bulky, nonpolar residues with alanine, which we refer to as \"alanization\". This allows the ligand to interact more closely with polar sidechains, which help to orient the ligand. Additionally, alanization reduces the impact of incorrect sidechain placement on ligand placement, a concern that sometimes requires user intervention. With DarwinDock working for standard small molecules, it was then necessary to modify the procedure to work on challenging GAG ligands, which are large and have strong negative charges. A modification to DarwinDock \u2013 GAG-Dock \u2013 allows the method to be applied to GAGs and protein surface interactions. GAGs are large, linear polysaccharides with strong negative charge. They typically interact with the surfaces of proteins, rather than the cavities favored by most small-molecule drugs. GAG-Dock systematically samples the protein surface for unknown binding sites and modifies the pose generation to allow for large, surface-interacting ligands. GAG-Dock allowed us to study several systems important for neuronal development and answer interesting questions posed by experiment. Finally, we needed a way to validate our predictions for GAG binding sites. We used a systematic approach to identify sets of beneficial mutations to the GAG binding sites by building up from individual in silico mutations. Standard mutation experiments typically employ large mutations, such as arginine to alanine, which decrease or destroy binding. However, such information is not always definitive, as large mutations can have wide-ranging effects beyond direct protein-ligand interactions. Mutations that increase binding, however, are less ambiguous because they must form new interactions with the ligand in order to affect binding energies or affinity. Therefore, we have identified and proposed sets of mutations for our GAG predictions for PTPs, NgR1, NgR3, and EphB3. We encourage our experimentalist colleagues to try these mutations and validate our predictions.
", "doi": "10.7907/Z91Z42GS", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10333", "collection": "thesis", "collection_id": "10333", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092017-125142341", "primary_object_url": { "basename": "Thesis11.pdf", "content": "final", "filesize": 121260400, "license": "other", "mime_type": "application/pdf", "url": "/10333/1/Thesis11.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Architecture of the Nuclear Pore Complex", "author": [ { "family_name": "Lin", "given_name": "Daniel Hanyang", "clpid": "Lin-Daniel-Hanyang" } ], "thesis_advisor": [ { "family_name": "Hoelz", "given_name": "Andre", "clpid": "Hoelz-A" } ], "thesis_committee": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hoelz", "given_name": "Andre", "clpid": "Hoelz-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nucleocytoplasmic transport, the regulated trafficking of macromolecules in and out of the nucleus, occurs primarily through nuclear pore complexes (NPCs). NPCs are massive macromolecular machines embedded in the nuclear envelope which generate ~40 nanometer transport channels to facilitate transport. Because of its size and complexity (~1000 subunits, ~120 MDa), the structure of the NPC has remained poorly understood. This thesis presents a bottom-up approach to understanding the structure and function of the NPC through reconstitution of the proteins and structural and biochemical studies. The first three chapters present work towards determining the composite structure of the symmetric core of the NPC. X-ray crystal structures are described for many of the components of the symmetric core. This includes a heterohexameric coat nucleoporin complex containing Nup120, Nup85, Nup145C, Sec13, Seh1, and Nup84, revealing how these proteins assemble into one of the main subcomplexes in the NPC. Reconstitution of the symmetric core components and analysis of the protein-protein interaction between the components provides a detailed biochemical map for the protein interaction network in the NPC. X-ray crystal structures of overlapping fragments facilitate the generation of accurate atomic model for full-length proteins. An iterative, sequential docking approach is developed to dock these models into a cryoelectron tomographic reconstruction of the human NPC, yielding a composite model for the structure of the symmetric core of the NPC. In the next two chapters, this analysis is extended to the cytoplasmic-specific decorations of the NPC. The structure of the C-terminal domain of Nup358 is reported and its catalytic activity is described. Lastly, reconstitution of human DDX19 activation by the NPC reveals mechanistic insight into how the NPC directly regulates the last step of mRNA export.", "doi": "10.7907/Z98P5XK8", "publication_date": "2017-06-16", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10227", "collection": "thesis", "collection_id": "10227", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302017-225018783", "type": "thesis", "title": "Biophysical Studies of Ligand-gated Ion Channels", "author": [ { "family_name": "Wong", "given_name": "Betty Ko", "clpid": "Wong-Betty-Ko" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Gradinaru", "given_name": "Viviana", "clpid": "Gradinaru-V" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation describes building a methodology for and the biophysical studies of ligand-gated ion channels (LGICs).
\r\n\r\nThe primary focus of the first half of this dissertation is on developing a fluorescence-based assay to broadly study LGICs. Chapter 2 describes the site-selective incorporation of a turn-on fluorophore via unnatural amino acid mutagenesis on the mouse muscle-type nicotinic acetylcholine receptor (nAChR) in Xenopus oocytes as a proof-of-principle study. This method has proven to yield very low levels of undesired fluorescent background, which was a problem for previous incorporation techniques. Chapter 3 describes efforts towards imaging this in vivo system using lifetime imaging with efforts hampered by the inability to detect a clear signal. Chapter 4 describes efforts to apply the lifetime imaging approach towards a different system involving 5-HT3 proteins fused to fluorescent proteins in COS-7 cells.
\r\n\r\nThe second half of this dissertation focuses on studies of menthol, a flavorant added to cigarettes that contributes to smoking addiction, as a negative allosteric modulator of the \u03b1\u03b242 nAChR. Chapter 5 reveals the stereochemical effects, or rather lack of, of menthol on the two stoichiometries of the \u03b1\u03b242 receptor. Chapter 6 seeks to identify the residue interactions with menthol of the \u03b1\u03b242 receptor using a combination of computational and experimental studies.
\r\n", "doi": "10.7907/Z9TT4P0Q", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10175", "collection": "thesis", "collection_id": "10175", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05162017-130844945", "type": "thesis", "title": "Structure and Function of the Mycobacterial Mechanosensitive Channel of Large Conductance, MscL\r ", "author": [ { "family_name": "Herrera", "given_name": "Nadia", "orcid": "0000-0003-4157-9429", "clpid": "Herrera-Nadia" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "MscL is a ubiquitous channel found in bacterial membranes. It provides a protective response to osmotic downshock by opening and closing in response to tension in the membrane. A number of studies have aimed to develop a mechanism for the gating of MscL in E. coli, but structural details describing the process have remained elusive. A few structures of non-conducting states of MscL have been solved using X-ray crystallography, Mycobacterium tuberculosis (Mt) MscL and Staphylococcus aureus (Sa) MscL with a C-terminal domain truncation. In addition, the structure of the E. coli (Ec) MscL C-terminal cytoplasmic domain has been solved.
\r\n \r\nThe goals of the studies presented in this thesis are as follows: (i) capturing a C-terminal domain truncation of MtMscL using X-ray crystallography, and (ii) analyzing the functional regulation of MscL channels in mycobacteria. To achieve the latter goal, we generated a knockout of the mscL gene in a fast-growing mycobacteria species, Mycobacterium smegmatis. This strain was used to analyze the role of MscL in the cell during antibiotic entry. Structural studies of MtMscL are focused on identifying the role of the C-terminal domain by studying a channel with a truncation at the C-terminal domain. The motivation for this goal comes from the structure of SaMscL, which showed that truncation of the C-terminal domain resulted in crystallizing the protein as a tetramer, an alternative oligomeric state to the pentameric state observed for the MtMscL structure. Studies on an MtMscL C-terminal domain truncation aimed to further establish that correlation. This protein was overexpressed in E. coli BL21 DE3 mscL-, purified, and crystallized by sitting drop vapor diffusion. Native crystals diffracted to 6.5 \u00c5, and heavy atom derivative crystals diffracted to 5.8 \u00c5. The structure of the MtMscL C-terminal truncation has been solved, and is presented in this thesis. Our studies on the structure show that the pentameric state of the channel remains intact upon truncation of the C-terminal domain. To analyze the function of our mutant, we utilized patch clamp electrophysiology studies using our expression strain as the giant spheroplast platform. The findings from the electrophysiology studies indicate that MtMscL C-terminal domain truncation results in a channel that has gating tension requirements similar to EcMscL, whereas full-length MtMscL has much higher gating tension requirements than our construct. In addition, the role of MscL in mycobacterial antibiotic susceptibility is being tested in Mycobacterium smegmatis. We have created a strain of M. smegmatis with the mscL gene knocked out, MC2155 mscL- and we have observed that upon deletion of mscL an increase in tolerance to spectinomycin is observed in our knockout strain.
", "doi": "10.7907/Z9JH3J77", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10005", "collection": "thesis", "collection_id": "10005", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062017-172749049", "primary_object_url": { "basename": "Rosebrugh_Lauren_2017_Thesis.pdf", "content": "final", "filesize": 27455021, "license": "other", "mime_type": "application/pdf", "url": "/10005/1/Rosebrugh_Lauren_2017_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stereoselective Olefin Metathesis Processes Using Cyclometalated Ruthenium Alkylidene Complexes", "author": [ { "family_name": "Rosebrugh", "given_name": "Lauren Estelle", "clpid": "Rosebrugh-Lauren-Estelle" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The recent development of a class of Z-selective ruthenium metathesis catalysts containing a crucial cyclometalated N-heterocyclic carbene (NHC) ligand has extended the applicability of ruthenium-mediated olefin metathesis to the production of a variety of useful Z-olefin-containing small molecules, polymers, and natural products. This thesis explores the synthesis and application of a number of novel Z-selective cyclometalated ruthenium alkylidene complexes displaying enhanced activity and selectivity across a range of metathesis transformations. Mechanistic investigations aimed at understanding and controlling stereoselectivity specifically in ring-opening metathesis polymerization (ROMP) are also detailed.
\r\n\r\nChapter 2 describes the preparation of new Z-selective cyclometalated ruthenium metathesis catalysts via an improved method employing sodium carboxylates. Effects of the cyclometalated NHC ligand on catalyst activity and selectivity in several cross metathesis assays, as well as macrocyclic ring-closing metathesis and other industrially relevant transformations, are investigated.
\r\n\r\nChapter 3 relates a story in two parts: the first details the synthesis and activity of a series of novel cyclometalated ruthenium alkylidenes displaying unprecedented cis,syndio-selectivity in the ROMP of norbornene- and norbornadiene-derived monomers. The second comprises an extensive study into the origins of stereoselectivity in ROMP in these and related cyclometalated ruthenium initiators. Experimental results are used in conjunction with a computational analysis of propagation transition states to develop a complete stereochemical model for cis,syndio-selctivity in these systems.
", "doi": "10.7907/Z9C53HVW", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10329", "collection": "thesis", "collection_id": "10329", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092017-062335915", "type": "thesis", "title": "Targeting DNA Mismatches with Luminescent Ruthenium Complexes", "author": [ { "family_name": "Boynton", "given_name": "Adam Nathaniel", "clpid": "Boynton-Adam-Nathaniel" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA base pair mismatches occur naturally in cells, typically as a result of errors during replication. Cells have evolved a DNA damage response pathway called mismatch repair (MMR) that identifies and corrects base pair mismatches in newly synthesized DNA. However, proteins involved in MMR can undergo mutations, rendering them incapable of correcting mismatches. Such deficiencies in MMR leads to an increase in genetic mutations and are associated with several forms of cancer. Because a higher mismatch frequency serves as an early indicator of cancer progression, DNA mismatches are a promising target in the design of small molecule therapeutics and diagnostics. In this context, transition metal complexes are prime candidates, owing to their\u00a0valuable spectroscopic and photophysical properties and versatile coordination sphere geometries. Our laboratory focuses on generating octahedral rhodium and ruthenium complexes that selectively target DNA mismatches. A class of rhodium complexes bearing sterically expansive planar ligands bind DNA mismatches with high selectivity and exhibit preferential cytotoxicity towards MMR-deficient cancer cells. These compounds bind to DNA through metalloinsertion, in which the bulky ligand inserts into the duplex at the thermodynamically destabilized mismatch site, displacing the mismatched bases into the DNA groove.
\r\n\r\nHerein we describe recent advances in the development of luminescent ruthenium complexes that selectively probe DNA mismatches. We demonstrate that [Ru(Me4phen)2(dppz)]2+ (Me4phen = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2\u2019,3\u2019-c]phenazine) is a DNA \u201clight switch\u201d that exhibits a significantly brighter steady-state emission in the presence of a DNA duplex containing a mismatch relative to completely well-matched DNA. Importantly, the bulky Me4phen ancillary ligands discourage deep intercalation of dppz between well-matched base pairs, and instead, [Ru(Me4phen)2(dppz)]2+ favors metalloinsertion at thermodynamically destabilized mismatches. [Ru(Me4phen)2(dppz)]2+ possesses a higher binding affinity towards a DNA mismatch relative to well-matched base pairs, and furthermore exhibits a longer excited-state emission lifetime when bound to a mismatch compared to that when intercalated at well-matched sites; both of these observations contribute to the dramatic steady-state emission enhancement detected with the mismatched DNA duplex. Additionally, we reveal that the right-handed delta (\u2206) isomer of [Ru(Me4phen)2(dppz)]2+ is the enantiomer which imparts all mismatch selectivity, consistent with the handedness of B-form DNA.
\r\n\r\nAnother mismatch-specific luminescent probe presented in this work is [Ru(bpy)2(BNIQ)]2+ (bpy = 2,2\u2019-bipyridine; BNIQ = benzo[c][1,7]naphthyridine-1-isoquinoline). In contrast to [Ru(Me4phen)2(dppz)]2+, the BNIQ complex exploits a bulky inserting ligand that selectively undergoes metalloinsertion at a DNA mismatch. This compound too exhibits a brighter steady-state emission in the presence of a mismatched duplex compared to entirely well-matched DNA, which we attribute to the fact that [Ru(bpy)2(BNIQ)]2+ possesses nearly a 500-fold higher binding affinity for the mismatch site compared to well-matched base pairs. Taken together, [Ru(Me4phen)2(dppz)]2+ and [Ru(bpy)2(BNIQ)]2+ represent two different yet valid approaches in the rational design of mismatch-specific small molecules, one based on ancillary ligand functionalization and the other on incorporating a sterically expansive inserting ligand.
\r\n\r\nA third approach towards the design of mismatch-specific luminescent ruthenium probes that is briefly explored here is the modification of the intercalating dppz ligand of [Ru(bpy)2(dppz)]2+. Bearing a dppz ligand substituted with four methyl groups, [Ru(bpy)2(tmdppz)]2+ (tmdppz = 3,4,7,8-tetramethyl dipyridophenazine) shows no luminescence discrimination between mismatched and well-matched duplexes. This observation ostensibly arises from the fact that the appended methyl groups shield the dppz phenazine nitrogen atoms from interactions with water when intercalated within the DNA.
\r\n\r\nWith mismatch-specific luminescent metalloinsertors such as [Ru(Me4phen)2(dppz)]2+ in hand, we have commenced biological investigations to see whether these compounds can serve as luminescent proxies for rhodium metalloinsertors in MMR-deficient cancer cells. Confocal microscopy of HCT116N and HCT116O cells reveals that [Ru(Me4phen)2(dppz)]2+ does preferentially localize to mitochondria, unlike potent cell-selective rhodium complexes such as [Rh(chrysi)(phen)(PPO)]2+ (PPO = 2-(pyridine-2-yl)propan-2-ol; chrysi = 5,6-chrysenequinone diimine); however, [Ru(Me4phen)2(dppz)]2+ shows some degree of nuclear entry. Here our goal is the application of the mismatch-specific luminescent probe in co-localization experiments to investigate what proteins are involved in the DNA damage response that is activated upon metalloinsertor binding in cellulo.
\r\n\r\nThe work presented here expands beyond the study of luminescent ruthenium complexes. Amino acid conjugates of the earlier-generation rhodium metalloinsertor [Rh(HDPA)2(chrysi)]3+ (HDPA = 2,2\u2019-dipyridylamine) were synthesized. While these conjugates exhibit mismatch binding affinities comparable to other rhodium metalloinsertors, they lose cell-selective biological activity, which may arise from altered uptake and/or sub-cellular localization. Finally, preliminary investigations were conducted on [Re(CO)3(pyOEt)(dppn)]+ (pyOEt = ethyl 3-(pyridin-4-yl)propanoate; dppn = benzodipyridophenazine) and [Ru(CN)(tpy)(dppz)]+ (tpy = terpyridine; CN = cyano), which were designed as IR-active probes to study the kinetics of DNA-mediated charge transport (CT) by time-resolved infrared (TRIR) spectroscopy. While these complexes do not possess the desired spectral TRIR properties as originally intended, steady-state luminescence experiments do suggest that this donor-acceptor pair is capable of undergoing DNA-mediated electron transfer.
\r\n\r\nAltogether, this work demonstrates the versatility of transition metal complexes as non-covalent probes for DNA. Importantly, through the rational modification of their three-dimensional ligand scaffold, one can achieve site-specific recognition of clinically relevant biomarkers such as DNA mismatches.
", "doi": "10.7907/Z9CF9N5M", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9891", "collection": "thesis", "collection_id": "9891", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07182016-144756449", "primary_object_url": { "basename": "Aron_Kamajaya_2016_thesis_final.pdf", "content": "final", "filesize": 7119497, "license": "other", "mime_type": "application/pdf", "url": "/9891/1/Aron_Kamajaya_2016_thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Structural Study of Piezo Channel, a Unique Family of Eukaryotic Mechanosensitive Channel", "author": [ { "family_name": "Kamajaya", "given_name": "Aron", "clpid": "Kamajaya-Aron" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Piezo is a unique family of eukaryotic mechanosensitive (MS) channel. With over 2500 amino acids per subunit, intact Piezo channel is one of the largest ion channels known to date. Two versions of Piezo can be found in vertebrates, namely PIEZO1 and PIEZO2. PIEZO1 appears to play roles in processes which control physiological homeostasis, whereas PIEZO2 assumes roles in mechanical somatosensation. A number of mutations mapped onto PIEZO1 or PIEZO2 are found in several hereditary human diseases, such as Dehydrated Hereditary Stomatocytosis, Gordon syndrome, and Distal Arthrogryposis. Although biochemical and functional studies provided many insightful findings, structural study of Piezo was very minimal. Herein, I described the structural investigation of Piezo channel. In the first study, we isolated a conserved soluble domain of Piezo (C-terminal loop 2, CTL2) from the C. elegans homolog, and provided the first molecular glimpse into this enigmatic MS channel. Subsequently, I described challenges that are associated with the expression and protein preparation of the full length Piezo channel. Recently, the full length mouse PIEZO1 structure solved by single particle cryo-EM revealed trimeric arrangement of the intact channel. CTL2 domain forms an extracellular cap which makes up the central core in this Piezo model. Lastly, we isolated a stable C-terminal fragment of Piezo. This fragment corresponds to the entire central core of Piezo channel and a few upstream transmembrane helices. This fragment can be localized to the plasma membrane. Further investigation is needed to look at the functionality of this fragment.", "doi": "10.7907/Z9JQ0Z00", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10166", "collection": "thesis", "collection_id": "10166", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112017-233327193", "type": "thesis", "title": "Non-Canonical Amino Acid Mutagenesis of Position B28 In Insulin with Proline Analogs", "author": [ { "family_name": "Lieblich", "given_name": "Seth Aharon", "clpid": "Lieblich-Seth-Aharon" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Insulin is a protein hormone that is crucial for maintaining the concentration of blood glucose in vivo and is used clinically as a drug for the treatment of diabetes.
\r\n\r\nChapter I provides for an overview and background on the state of the art in insulin treatment of diabetes and the many attempts, over 95 years, to improve the pharmaceutically relevant properties of insulin and improve our understanding of the model globular protein.
\r\n\r\nChapter II demonstrates the incorporation of hydroxyproline analogs into insulin and shares the discovery of insulin with enhanced stability and an accelerated kinetic rate of dissociation. We also provide the highest resolution structure deposited in the PDB of insulin in the T2 state and the 3rd highest of any insulin to date.
\r\n \r\nChapter III extends the incorporation of proline analogs in insulin to include fluorinated insulins. We also provide, for the first time, high-resolution structures of a single globular protein systematically mutated with all possible stereoisomers of fluorination at the 4-position on a single proline residue (4S, 4R, di-substituted).
\r\n\r\nChapter IV extends the incorporation of proline analogs in insulin to include ring variant analogs. We also provide, for the first time, high-resolution structures of globular proteins containing pipecolic acid, azetidine-2-carboxylic acid and 3,4 dehydroproline in the polypeptide chain.
\r\n\r\nChapter V discusses the significance of the findings described herein and discusses future directions to undertake in further engineering insulin for improved characteristics.
\r\n\r\nThis thesis describes a systematic approach, akin to medicinal chemistry, of altering a particular protein side chain by atomistic changes. I hope that the breadth of different amino acids incorporated into a single globular protein combined with the structural, functional, thermodynamic and kinetic information contained within this set of mutants will provide future protein engineers, computational protein designers and proline enthusiasts with a wealth of new information to be used to improve our understanding of proteins and predictive power.
\r\n", "doi": "10.7907/Z90Z71BD", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10164", "collection": "thesis", "collection_id": "10164", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112017-113214704", "primary_object_url": { "basename": "MorrisonChristine_2017_Thesis.pdf", "content": "final", "filesize": 9506224, "license": "other", "mime_type": "application/pdf", "url": "/10164/78/MorrisonChristine_2017_Thesis.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Insights into the Mechanism of Biological Nitrogen Fixation through Characterization of the Nitrogenase Molybdenum-Iron Protein", "author": [ { "family_name": "Morrison", "given_name": "Christine Nichole", "orcid": "0000-0002-4180-8407", "clpid": "Morrison-Christine-Nichole" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nitrogen fixation, the process of converting dinitrogen to ammonia, is performed industrially and biologically by the Haber-Bosch process and nitrogenase, respectively. The resulting ammonia is largely used as fertilizer. Since there is a finite amount of ammonia produced by nitrogenase, we are heavily dependent on the Haber-Bosch process \u2013 only two-fifths of the world\u2019s population could be fed without it. Although the importance of the Haber-Bosch process cannot be overstated, our dependence on it has several drawbacks, including significant energy costs (~5% of the annual natural gas consumption), greenhouse gas emissions, and nitrate runoffs. By understanding the biological mechanism of nitrogen fixation, we may be able to (1) develop more efficient nitrogen fixing catalysts to replace those in the Haber-Bosch process or (2) express de novo nitrogen fixing proteins in plants so crops can essentially fertilize themselves. The projects described in this thesis aim to contribute to our understanding of the mechanism of biological nitrogen fixation through structural studies of nitrogenase. Nitrogenase consists of the iron and molybdenum-iron (MoFe) proteins, the latter of which contains the active site, the FeMo-cofactor. Throughout my work, I compare the MoFe proteins from Azotobacter vinelandii (Av1) and Clostridium pasteurianum (Cp1), the two most structurally divergent molybdenum nitrogenases known. Determining the similarities and differences between these proteins may aid our understanding of biological nitrogen fixation. My first project (Chapter III) compares a 1.08 \u00c5 Cp1 X-ray structure to a previously published 1.0 \u00c5 Av1 structure. I determined that the center atom of the Cp1 FeMo-cofactor is carbon, showing conservation of cofactor structure among molybdenum nitrogenases. Next, I compared substrate pathways in Av1 and Cp1 via Xe pressurization and identification of small molecule binding sites (Chapter IV). My most significant results include the structural and electronic characterization of a reversible protonated resting state of Av1 and Cp1 (Chapter VII).
", "doi": "10.7907/Z95B00HX", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10139", "collection": "thesis", "collection_id": "10139", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04172017-185314743", "type": "thesis", "title": "Structures and Reactions of Diplatinum Complexes", "author": [ { "family_name": "Darnton", "given_name": "Tania Victoria", "clpid": "Darnton-Tania-Victoria" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A d8\u2212d8 complex [Pt2(\u03bc-P2O5(BF2)4]4\u2212 (abbreviated Pt(pop-BF2)4\u2212) undergoes two 1e\u2212 reductions at E1/2 = \u22121.68 and Ep = \u22122.46 V (vs Fc+/Fc) producing reduced Pt(pop-B2)5\u2212 and superreduced Pt(pop-BF2)6\u2212 species, respectively. The EPR spectrum of Pt(pop-BF2)5\u2212 and UV\u2212vis spectra of both the reduced and the superreduced complexes, together with TD-DFT calculations, reveal successive filling of the 6p\u03c3 orbital accompanied by gradual strengthening of Pt\u2212Pt bonding interactions and, because of 6p\u03c3 delocalization, of Pt\u2212P bonds in the course of the two reductions. Both reduction steps proceed without changing either d8 Pt electronic configuration, making the superreduced Pt(pop-BF2)6\u2212 a very rare 6p2 \u03c3-bonded binuclear complex. However, the Pt\u2212Pt \u03c3 bonding interaction is limited by the relatively long bridging-ligand-imposed Pt\u2212Pt distance accompanied by repulsive electronic congestion. Pt(pop-BF2)4\u2212 is predicted to be a very strong photooxidant (potentials of +1.57 and +0.86 V are estimated for the singlet and triplet d\u03c3*p\u03c3 excited states, respectively).
\r\n\r\nFurther study of the electronic excited states of Pt(pop-BF2)4- in the presence of luminescence quenchers revealed Stern-Volmer type dynamic quenching of the triplet state by trialkyl and triaryl amines. Quenching of the singlet as well as the triplet was observed in the presence of CoII trisbipyridine complexes, but sample decomposition and the observed presence of simultaneous static and dynamic quenching behaviors hampered quantitative analysis.
\r\n", "doi": "10.7907/Z9NK3C2J", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9270", "collection": "thesis", "collection_id": "9270", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11042015-142900908", "type": "thesis", "title": "Biochemical and Biophysical Characterization of Huntingtin", "author": [ { "family_name": "Owens", "given_name": "Gwen Ellen", "orcid": "0000-0003-0793-1994", "clpid": "Owens-Gwen-Ellen" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Huntington\u2019s disease (HD) is a fatal autosomal dominant neurodegenerative disease. HD has no cure, and patients pass away 10-20 years after the onset of symptoms. The causal mutation for HD is a trinucleotide repeat expansion in exon 1 of the huntingtin gene that leads to a polyglutamine (polyQ) repeat expansion in the N-terminal region of the huntingtin protein. Interestingly, there is a threshold of 37 polyQ repeats under which little or no disease exists; and above which, patients invariably show symptoms of HD. The huntingtin protein is a 350 kDa protein with unclear function. As the polyQ stretch expands, its propensity to aggregate increases with polyQ length. Models for polyQ toxicity include formation of aggregates that recruit and sequester essential cellular proteins, or altered function producing improper interactions between mutant huntingtin and other proteins. In both models, soluble expanded polyQ may be an intermediate state that can be targeted by potential therapeutics.
\r\n\r\nIn the first study described herein, the conformation of soluble, expanded polyQ was determined to be linear and extended using equilibrium gel filtration and small-angle X-ray scattering. While attempts to purify and crystallize domains of the huntingtin protein were unsuccessful, the aggregation of huntingtin exon 1 was investigated using other biochemical techniques including dynamic light scattering, turbidity analysis, Congo red staining, and thioflavin T fluorescence. Chapter 4 describes crystallization experiments sent to the International Space Station and determination of the X-ray crystal structure of the anti-polyQ Fab MW1. In the final study, multimeric fibronectin type III (FN3) domain proteins were engineered to bind with high avidity to expanded polyQ tracts in mutant huntingtin exon 1. Surface plasmon resonance was used to observe binding of monomeric and multimeric FN3 proteins with huntingtin.
", "doi": "10.7907/Z9TM7835", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9039", "collection": "thesis", "collection_id": "9039", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06292015-212932175", "primary_object_url": { "basename": "Martinez_Thomas_2016_Thesis.pdf", "content": "final", "filesize": 29641943, "license": "other", "mime_type": "application/pdf", "url": "/9039/1/Martinez_Thomas_2016_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Investigations of Pyrrole-Imidazole Polyamide Effects on DNA Replication", "author": [ { "family_name": "Martinez", "given_name": "Thomas Farid", "orcid": "0000-0002-4011-8164", "clpid": "Martinez-Thomas-Farid" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Pyrrole\u2013Imidazole polyamides are programmable, cell-permeable small molecules that bind in the minor groove of double-stranded DNA sequence-specifically. Polyamide binding has been shown to alter the local helical structure of DNA, disrupt protein-DNA interactions, and modulate endogenous gene expression. Py\u2013Im polyamides targeted to the androgen receptor-DNA interface have been observed to decrease expression of androgen-regulated genes, upregulate p53, and induce apoptosis in a hormone-sensitive prostate cancer cell line. Here we report that androgen response element (ARE)-targeted polyamides induced DNA replication stress in a hormone-insensitive prostate cancer cell line. The ATR checkpoint kinase was activated in response to this stress, causing phosphorylation of MCM2, and FANCD2 was monoubiquitinated. Surprisingly, little single-stranded DNA was exhibited, and the ATR targets RPA2 and Chk1 were not phosphorylated. We conclude that polyamide induces relatively low level replication stress, and suggest inhibition of the replicative helicase as a putative mechanism based on in vitro assays. We also demonstrate polyamide-induced inhibition of DNA replication in cell free extracts from X. laevis oocytes. In this system, inhibition of chromatin decondensation is observed, preventing DNA replication initiation. Finally, we show that Py-Im polyamides targeted to the ARE and ETS binding sequence downregulate AR- and ERG-driven signaling in a prostate cancer cell line harboring the TMPRSS2-ERG fusion. In a mouse xenograft model, ARE-targeted polyamide treatment reduced growth of the tumor.
", "doi": "10.7907/Z9RF5RZ2", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9133", "collection": "thesis", "collection_id": "9133", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09012015-173735253", "type": "thesis", "title": "The Molecular Basis of Lysine Acetylation: Addition, Removal, and Recognition", "author": [ { "family_name": "Davenport", "given_name": "Andrew M.", "clpid": "Davenport-Andrew-M" } ], "thesis_advisor": [ { "family_name": "Hoelz", "given_name": "Andre", "clpid": "Hoelz-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Hoelz", "given_name": "Andre", "clpid": "Hoelz-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Acetyltransferases and deacetylases catalyze the addition and removal, respectively, of acetyl groups to the epsilon-amino group of protein lysine residues. This modification can affect the function of a protein through several means, including the recruitment of specific binding partners called acetyl-lysine readers. Acetyltransferases, deacetylases, and acetyl-lysine readers have emerged as crucial regulators of biological processes and prominent targets for the treatment of human disease. This work describes a combination of structural, biochemical, biophysical, cell-biological, and organismal studies undertaken on a set of proteins that cumulatively include all steps of the acetylation process: the acetyltransferase MEC-17, the deacetylase SIRT1, and the acetyl-lysine reader DPF2. Tubulin acetylation by MEC-17 is associated with stable, long-lived microtubule structures. We determined the crystal structure of the catalytic domain of human MEC-17 in complex with the cofactor acetyl-CoA. The structure in combination with an extensive enzymatic analysis of MEC-17 mutants identified residues for cofactor and substrate recognition and activity. A large, evolutionarily conserved hydrophobic surface patch distal to the active site was shown to be necessary for catalysis, suggesting that specificity is achieved by interactions with the alpha-tubulin substrate that extend outside of the modified surface loop. Experiments in C. elegans showed that while MEC-17 is required for touch sensitivity, MEC-17 enzymatic activity is dispensible for this behavior. SIRT1 deacetylates a wide range of substrates, including p53, NF-kappaB, FOXO transcription factors, and PGC-1-alpha, with roles in cellular processes ranging from energy metabolism to cell survival. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an apo form and in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a beta-hairpin structure that complements the beta-sheet of the NAD^+-binding domain, covering an essentially invariant, hydrophobic surface. A comparison of the apo and cofactor bound structures revealed conformational changes throughout catalysis, including a rotation of a smaller subdomain with respect to the larger NAD^+-binding subdomain. A biochemical analysis identified key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain. DPF2 represses myeloid differentiation in acute myelogenous leukemia. Finally, we solved the crystal structure of the tandem PHD domain of human DPF2. We showed that DPF2 preferentially binds H3 tail peptides acetylated at Lys14, and binds H4 tail peptides with no preference for acetylation state. Through a structural and mutational analysis we identify the molecular basis of histone recognition. We propose a model for the role of DPF2 in AML and identify the DPF2 tandem PHD finger domain as a promising novel target for anti-leukemia therapeutics.", "doi": "10.7907/Z9BG2KWK", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9064", "collection": "thesis", "collection_id": "9064", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07212015-103405937", "type": "thesis", "title": "Physical, Metabolic, and Energetic Investigations of Methane-Metabolizing Microbial Communities", "author": [ { "family_name": "Marlow", "given_name": "Jeffrey James", "clpid": "Marlow-Jeffrey-James" } ], "thesis_advisor": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "orcid": "0000-0002-5374-6178", "clpid": "Orphan-V-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Fischer", "given_name": "Woodward W.", "orcid": "0000-0002-8836-3054", "clpid": "Fischer-W-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Hoehler", "given_name": "Tori M.", "clpid": "Hoehler-T-M" } ], "local_group": [ { "literal": "div_gps" } ], "abstract": "Understanding the roles of microorganisms in environmental settings by linking phylogenetic identity to metabolic function is a key challenge in delineating their broad-scale impact and functional diversity throughout the biosphere. This work addresses and extends such questions in the context of marine methane seeps, which represent globally relevant conduits for an important greenhouse gas. Through the application and development of a range of culture-independent tools, novel habitats for methanotrophic microbial communities were identified, established settings were characterized in new ways, and potential past conditions amenable to methane-based metabolism were proposed. Biomass abundance and metabolic activity measures \u2013 both catabolic and anabolic \u2013 demonstrated that authigenic carbonates associated with seep environments retain methanotrophic activity, not only within high-flow seep settings but also in adjacent locations exhibiting no visual evidence of chemosynthetic communities. Across this newly extended habitat, microbial diversity surveys revealed archaeal assemblages that were shaped primarily by seepage activity level and bacterial assemblages influenced more substantially by physical substrate type. In order to reliably measure methane consumption rates in these and other methanotrophic settings, a novel method was developed that traces deuterium atoms from the methane substrate into aqueous medium and uses empirically established scaling factors linked to radiotracer rate techniques to arrive at absolute methane consumption values. Stable isotope probing metaproteomic investigations exposed an array of functional diversity both within and beyond methane oxidation- and sulfate reduction-linked metabolisms, identifying components of each proposed enzyme in both pathways. A core set of commonly occurring unannotated protein products was identified as promising targets for future biochemical investigation. Physicochemical and energetic principles governing anaerobic methane oxidation were incorporated into a reaction transport model that was applied to putative settings on ancient Mars. Many conditions enabled exergonic model reactions, marking the metabolism and its attendant biomarkers as potentially promising targets for future astrobiological investigations. This set of inter-related investigations targeting methane metabolism extends the known and potential habitat of methanotrophic microbial communities and provides a more detailed understanding of their activity and functional diversity.", "doi": "10.7907/Z9W66HPS", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9762", "collection": "thesis", "collection_id": "9762", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05262016-050606547", "type": "thesis", "title": "Structural and Biochemical Characterization of Ligand Bound States of the FeMo-Cofactor of Nitrogenase", "author": [ { "family_name": "Perez", "given_name": "Kathryn A.", "clpid": "Perez-Kathryn-A" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nitrogenase is the only known enzyme capable of nitrogen fixation, the reduction of dinitrogen to ammonia, a metabolically available form of nitrogen. Developing an understanding of the complex mechanism required for biological nitrogen fixation requires that the enzyme be characterized in catalytically relevant states, such as those involving ligand binding and reduction. Nitrogenase catalyzes this reaction through the cyclic interaction of two metalloproteins, the Fe-protein and the MoFe-protein which contain three distinct metalloclusters, in an ATP-hydrolysis dependent electron transfer reaction. The binding and subsequent reduction of substrates requires multiple electrons donated from the Fe-protein to the MoFe-protein, in which the active site is located. In this study, we have structurally characterized the binding of two inhibitors to the FeMo-cofactor, CO and the Se of SeCN-. Both interactions involve the displacement of a single S, and the Se was used as a label to follow the interchange of three S sites within the FeMo-cofactor during catalysis. These finding change any future approaches to characterize the mechanism of biological nitrogen fixation, requiring that structural changes be considered for substrate binding and reduction.", "doi": "10.7907/Z9M043DX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9753", "collection": "thesis", "collection_id": "9753", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05252016-131059745", "primary_object_url": { "basename": "0_HSegal_Thesis_Final_Post Proof Reader.pdf", "content": "final", "filesize": 5688249, "license": "other", "mime_type": "application/pdf", "url": "/9753/4/0_HSegal_Thesis_Final_Post Proof Reader.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Electrochemical Methods to Study Iron-Sulfur Cluster Proteins", "author": [ { "family_name": "Segal", "given_name": "Helen Muriel", "clpid": "Segal-Helen-Muriel" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Electron transfer between proteins is an important mechanism in multiple biological processes. In this thesis, methods were developed to study electron transfer in two biological contexts: 1) DNA-mediated signaling between DNA binding proteins with 4Fe-4S clusters and 2) nitrogenase.
\r\n\r\nThe first portion of this thesis focuses on the spectroscopic and electrochemical characterization of the iron-sulfur cluster in Dna2. Dna2 is a helicase-nuclease that is involved in Okazaki fragment maturation, double strand break repair, mitochondrial genome maintenance, and telomere maintenance. Dna2 is one of multiple DNA repair and replication proteins that contain a 4Fe-4S cluster, a cofactor that generally participates in electron transfer processes. It has been proposed that these enzymes may use their 4Fe-4S clusters to signal one another over large molecular distances to coordinate their activity on biological time scales through DNA-mediated redox chemistry. A combination of EPR and UV-visible absorption spectroscopy along with electrochemistry studies on DNA-modified gold electrodes was performed to provide insight into the chemical characteristics of the 4Fe-4S cluster in Dna2. These studies also provide a foundation for how DNA charge transport might coordinate the action of eukaryotic DNA repair and replication proteins with 4Fe-4S clusters.
\r\n\r\nThe second portion of this thesis describes the development of electrochemical methods to study nitrogenase, the enzyme that catalyzes the reduction of atmospheric dinitrogen to bioavailable ammonia. First, flavodoxin II, the biological reductant of the Fe-protein of nitrogenase, was characterized using a combination of electrochemical and structural methods to determine the molecular interactions that facilitate reduction of the nitrogenase iron protein. Second, two electrochemical methods, edge-plane pyrolytic graphite electrodes and single crystal gold electrodes modified with \u03c9-functionalized alkane-thiols, were adapted to study the redox chemistry at the iron-sulfur cluster of the Fe-protein. These studies provided insight into both the fundamental characteristics of electron transfer reactions involving nitrogenase, as well as insight into how to better study this enzyme using electrochemical methods.
\r\n\r\n", "doi": "10.7907/Z92Z13HT", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9063", "collection": "thesis", "collection_id": "9063", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07192015-214603085", "primary_object_url": { "basename": "DNA CT signaling within the cell_MAG_2015.pdf", "content": "final", "filesize": 4584274, "license": "other", "mime_type": "application/pdf", "url": "/9063/1/DNA CT signaling within the cell_MAG_2015.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Signaling Within the Cell", "author": [ { "family_name": "Grodick", "given_name": "Michael Andrew", "clpid": "Grodick-Michael-Andrew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA possesses the curious ability to conduct charge longitudinally through the \u03c0-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.
\r\n\r\nHerein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.
\r\n\r\nIn separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?
\r\n\r\nUvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.
\r\n\r\n\r\n", "doi": "10.7907/Z9F769GX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9738", "collection": "thesis", "collection_id": "9738", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05232016-135949451", "type": "thesis", "title": "Mechanisms of Regulation and Fidelity in Tail-Anchored Membrane Protein Targeting", "author": [ { "family_name": "Rao", "given_name": "Meera", "orcid": "0000-0001-8650-6253", "clpid": "Rao-Meera" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Accurate protein localization is crucial to generate and to maintain cellular organization. Achieving accuracy is challenging, as the molecular signals that dictate a protein\u2019s destination are often promiscuous. The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at its extreme C-terminus, presents major challenges to protein targeting machineries. This dissertation explores how TA capture and release are spatially and temporally regulated in the Guided Entry of Tail Anchored proteins (GET) pathway and how endoplasmic reticulum (ER) destined TAs are targeted with high fidelity.
\r\n\r\nA quantitative framework of the Get3 ATPase cycle reveals that ATP and GET pathway effector proteins specifically induce multiple conformational changes in Get3, which culminate in the ATPase activation that drives unidirectional targeting in the pathway. The Get4/5 TA loading complex locks Get3 in the ATP-bound state that is primed for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction.
\r\n\r\nAdditional analyses define multiple physicochemical features that distinguish TA proteins destined to different organelles. The GET pathway selects for these features at distinct stages using mechanisms such as differential binding, induced fit, and kinetic proofreading after ATP hydrolysis by Get3. These results reveal new roles for the cochaperone Sgt2 in providing key selection filters, and provide a biological logic for the complex cascade of substrate relay events during post-translational membrane protein targeting.
", "doi": "10.7907/Z9G73BQN", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9299", "collection": "thesis", "collection_id": "9299", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12012015-124453213", "type": "thesis", "title": "Proton-Coupled Reduction of N\u2082 Facilitated by Molecular Fe Complexes", "author": [ { "family_name": "Rittle", "given_name": "Jonathan Daniel", "clpid": "Rittle-Jonathan-Daniel" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The activation of Fe-coordinated N2 via the formal addition of hydrogen atom equivalents is explored in this thesis. These reactions may occur in nitrogenase enzymes during the biological conversion of N2 to NH3. To understand these reactions, the N2 reactivity of a series of molecular Fe(N2) platforms is investigated. A trigonal pyramidal, carbon-ligated FeI complex was prepared that displays a similar geometry to that of the resting state 'belt' Fe atoms of nitrogenase. Upon reduction, this species was shown to coordinate N2, concomitant with significant weakening of the C-Fe interaction. This hemilability of the axial ligand may play a critical role in mediating the interconversion of Fe(NxHy) species during N2 conversion to NH3. In fact, a trigonal pyramidal borane-ligated Fe complex was shown to catalyze this transformation, generating up to 8.49 equivalents of NH3. To shed light on the mechanistic details of this reaction, protonation of a borane-ligated Fe(N2) complex was investigated and found to give rise to a mixture of species that contains an iron hydrazido(2-) [Fe(NNH2)] complex. The identification of this species is suggestive of an early N-N bond cleavage event en route to NH3 production, but the highly-reactive nature of this complex frustrated direct attempts to probe this possibility. A structurally-analogous silyl-ligated Fe(N2) complex was found to react productively with hydrogen atom equivalents, giving rise to an isolable Fe(NNH2) species. Spectroscopic and crystallographic studies benefited from the enhanced stability of this complex relative to the borane analogue. One-electron reduction of this species initiates a spontaneous disproportionation reaction with an iron hydrazine [Fe(NH2NH2)] complex as the predominant reaction product. This transformation provides support for an Fe-mediated N2 activation mechanism that proceeds via a late N-N bond cleavage. In hopes of gaining more fundamental insight into these reactions, a series of Fe(CN) complexes were prepared and reacted with hydrogen-atom equivalents. Significant quantities of CH4 and NH3 are generated in these reactions as a result of complete C-N bond activation. A series of Fe(CNHx) were found to be exceptionally stable and may be intermediates in these reactions. The stability of these compounds permitted collection of thermodynamic parameters pertinent to the unique N-H bonds. This data is comparatively discussed with the theoretically-predicted data of the N2-derived Fe(NNHx) species. Exceptionally-weak N-H bond enthalpies are found for many of these compounds, and sheds light on their short-lived nature and tendency to evolve H2. As a whole, these works both establish and provide a means to understand Fe-mediated N2 activation via the addition of hydrogen atom equivalents.", "doi": "10.7907/Z9QJ7F7D", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9716", "collection": "thesis", "collection_id": "9716", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05122016-095434020", "primary_object_url": { "basename": "Shafaat_OliverSyed_2016.pdf", "content": "final", "filesize": 35259152, "license": "other", "mime_type": "application/pdf", "url": "/9716/1/Shafaat_OliverSyed_2016.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Temporal Control of Ion Channel Activation", "author": [ { "family_name": "Shafaat", "given_name": "Oliver Syed", "orcid": "0000-0002-3170-2553", "clpid": "Shafaat-Oliver-Syed" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ion channels are a large class of integral membrane proteins that allow for the diffusion of ions across a cellular membrane and are found in all forms of life. Pentameric ligand-gated ion channels (pLGICs) comprise a large family of proteins that include the nicotinic acetylcholine receptor (nAChR) and the \u03b3-aminobutyric acid (GABA) receptor. These ion channels are responsible for the fast synaptic transmission that occurs in humans and as a result are of fundamental biological importance. pLGICs bind ligands (neurotransmitters), and upon ligand-binding undergo activation. The activation event causes an ion channel to enter a new physical state that is able to conduct ions. Ion channels allow for the flux of ions across the membrane through a pore that is formed upon ion channel activation. For pLGICs to function properly both ligand-binding and ion channel activation must occur. The ligand-binding event has been studied extensively over the past few decades, and a detailed mechanism of binding has emerged. During activation the ion channel must undergo structural rearrangements that allow the protein to enter a conformation in which ions can flow through. Despite this great and ubiquitous importance, a fundamental understanding of the ion channel activation mechanism and kinetics, as well as concomitant structural arrangements, remains elusive.
\r\n\r\nThis dissertation describes efforts that have been made to temporally control the activation of ligand-gated ion channels. Temporal control of ion channel activation provides a means by which to activate ion channels when desired. The majority of this work examines the use of light to activate ion channels. Several photocages were examined in this thesis; photocages are molecules that release a ligand under irradiation, and, for the work described here, the released ligand then activates the ion channel. First, a new water-soluble photoacid was developed for the activation\r\nof proton-sensitive ion channels. Activation of acid-sensing ion channels, ASIC2a and GLIC, was observed only upon irradiation. Next, a variety of Ru2+ photocages were also developed for the release of amine ligands. The Ru2+ systems interacted in a deleterious manner with a representative subset of biologically essential ion channels. The rapid mixing of ion channels with agonist was also examined. A detection system was built to monitor ion channels activation in the rapid mixing experiments. I have shown that liposomes, and functionally-reconstituted ELIC, are not destroyed during the mixing process. The work presented here provides the means to deliver agonist to ligand-gated ion channels in a controlled fashion.
\r\n\r\n", "doi": "10.7907/Z98P5XGX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8742", "collection": "thesis", "collection_id": "8742", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12152014-153232034", "primary_object_url": { "basename": "Idigo_2015.pdf", "content": "final", "filesize": 5222567, "license": "other", "mime_type": "application/pdf", "url": "/8742/21/Idigo_2015.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MsCL)", "author": [ { "family_name": "Idigo", "given_name": "Chinenye Abiodun", "clpid": "Idigo-Chinenye-Abiodun" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.
\r\n\r\nIt has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 \u00b0C \u2013 60 \u00b0C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.
\r\n\r\nExtensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.
\r\n\r\nMscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.
", "doi": "10.7907/Z9542KJ3", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:9007", "collection": "thesis", "collection_id": "9007", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062015-192618908", "primary_object_url": { "basename": "Anna Arnold_2015_Thesis full version.pdf", "content": "final", "filesize": 35527821, "license": "other", "mime_type": "application/pdf", "url": "/9007/143/Anna Arnold_2015_Thesis full version.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Protein Oxidation", "author": [ { "family_name": "Arnold", "given_name": "Anna Ruth", "clpid": "Arnold-Anna-Ruth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.
\r\n\r\nDps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.
\r\n\r\nFurther work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.
\r\n\r\nWe have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.
\r\n\r\nDNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.
\r\n\r\nWhile the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]3+ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.
\r\n\r\nIn conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.
", "doi": "10.7907/Z9ZW1HVN", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8927", "collection": "thesis", "collection_id": "8927", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-144036736", "primary_object_url": { "basename": "Christopher_Bruno_Marotta_2015-0522-Thesis.pdf", "content": "final", "filesize": 14106595, "license": "other", "mime_type": "application/pdf", "url": "/8927/1/Christopher_Bruno_Marotta_2015-0522-Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators", "author": [ { "family_name": "Marotta", "given_name": "Christopher Bruno", "orcid": "0000-0002-3110-0819", "clpid": "Marotta-Christopher-Bruno" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.
\r\n\t\r\nThe first half of this dissertation focuses on differential agonist selectivity of \u03b14\u03b22-containing nAChRs. The \u03b14\u03b22 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent \u03b14\u03b22 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a \u03b15\u03b14\u03b22 receptor-enriched population to test for a potential alternative agonist binding location at the \u03b15 \u03b14 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the \u03b15 subunit only incorporates at the accessory subunit location.
\r\n\t\r\nThe second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the \u03b17 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates \u03b17 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.
", "doi": "10.7907/Z9V122Q9", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8914", "collection": "thesis", "collection_id": "8914", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-064617250", "primary_object_url": { "basename": "Thesis_Alysia_Ahmed_052915.pdf", "content": "final", "filesize": 33527538, "license": "other", "mime_type": "application/pdf", "url": "/8914/1/Thesis_Alysia_Ahmed_052915.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structural Characterization of Pro-inflammatory and Anti-inflammatory Immunoglobulin G Fc Proteins", "author": [ { "family_name": "Ahmed", "given_name": "Alysia Ashley", "clpid": "Ahmed-Alysia-Ashley" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Immunoglobulin G (IgG) is central in mediating host defense due to its ability to target and eliminate invading pathogens. The fragment antigen binding (Fab) regions are responsible for antigen recognition; however the effector responses are encoded on the Fc region of IgG. IgG Fc displays considerable glycan heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Intravenous immunoglobulin G (IVIG) is pooled serum IgG from multiple donors and is used to treat individuals with autoimmune and inflammatory disorders such as rheumatoid arthritis and Kawasaki\u2019s disease, respectively. It contains all the subtypes of IgG (IgG1-4) and over 120 glycovariants due to variation of an Asparagine 297-linked glycan on the Fc. The species identified as the activating component of IVIG is sialylated IgG Fc. Comparisons of wild type Fc and sialylated Fc X-ray crystal structures suggests that sialylation causes an increase in conformational flexibility, which may be important for its anti-inflammatory properties.
\r\n\r\nAlthough glycan modifications can promote the anti-inflammatory properties of the Fc, there are amino acid substitutions that cause Fcs to initiate an enhanced immune response. Mutations in the Fc can cause up to a 100-fold increase in binding affinity to activating Fc gamma receptors located on immune cells, and have been shown to enhance antibody dependent cell-mediated cytotoxicity. This is important in developing therapeutic antibodies against cancer and infectious diseases. Structural studies of mutant Fcs in complex with activating receptors gave insight into new protein-protein interactions that lead to an enhanced binding affinity.
\r\n\r\nTogether these studies show how dynamic and diverse the Fc region is and how both protein and carbohydrate modifications can alter structure, leading to IgG Fc\u2019s switch from a pro-inflammatory to an anti-inflammatory protein.
", "doi": "10.7907/Z9GT5K33", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8728", "collection": "thesis", "collection_id": "8728", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11202014-184045649", "primary_object_url": { "basename": "20141029_Thesis_SS.pdf", "content": "final", "filesize": 33236461, "license": "other", "mime_type": "application/pdf", "url": "/8728/1/20141029_Thesis_SS.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Homo- and Heteronuclear Transition Metal Complexes Supported by Multinucleating Ligands", "author": [ { "family_name": "Suseno", "given_name": "Sandy", "clpid": "Suseno-Sandy" } ], "thesis_advisor": [ { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" } ], "thesis_committee": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation is mainly divided into two sub-parts: organometallic and bioinorganic/materials projects. The approach for the projects involves the use of two different multinucleating ligands to synthesize mono- and multinuclear complexes. Chapter 2 describes the synthesis of a multinucleating tris(phosphinoaryl)benzene ligand used to support mono-nickel and palladium complexes. The isolated mononuclear complexes were observed to undergo intramolecular arene C\u00ac\u2013H to C\u2013P functionalization. The transformation was studied by nuclear magnetic resonance spectroscopy and X-ray crystallography, and represents a rare type of C\u2013H functionalization mechanism, facilitated by the interactions of the group 10 metal with the arene \u03c0\u2013system.
\r\n\r\nChapter 3 describes the construction of multinickel complexes supported by the same triphosphine ligand from Chapter 2. This chapter shows how the central arene in the ligand\u2019s triarylbenzene framework can interact with dinickel and trinickel moieties in various binding modes. X-ray diffraction studies indicated that all compounds display strong metal\u2013arene interactions. A cofacial triangulo nickel(0) complex supported by this ligand scaffold was also isolated and characterized. This chapter demonstrates the use of an arene as versatile ligand design element for small molecular clusters.
\r\n\r\nChapter 4 presents the syntheses of a series of discrete mixed transition metal Mn oxido clusters and their characterization. The synthesis of these oxide clusters displaying two types of transition metals were targeted for systematic metal composition-property studies relevant to mixed transition metal oxides employed in electrocatalysis. A series of heterometallic trimanganese tetraoxido cubanes capped with a redox-active metal [MMn3O4] (M = Fe, Co, Ni, Cu) was synthesized starting from a [CaMn3O4] precursor and structurally characterized by X-ray crystallography and anomalous diffraction to conclusively determine that M is incorporated at a single position in the cluster. The electrochemical properties of these complexes were studied via cyclic voltammetry. The redox chemistry of the series of complexes was investigated by the addition of a reductant and oxidant. X-ray absorption and electron paramagnetic resonance spectroscopies were also employed to evaluate the product of the oxidation/reduction reaction to determine the site of electron transfer given the presence of two types of redox-active metals. Additional studies on oxygen atom transfer reactivities of [MMn3O4] and [MMn3O2] series were performed to investigate the effect of the heterometal M in the reaction rates.
\r\n\r\nChapter 5 focuses on the use of [CoMn3O4] and [NiMn3O4] cubane complexes discussed in Chapter 4 as precursors to heterogeneous oxygen evolution reaction (OER) electrocatalysts. These well-defined complexes were dropcasted on electrodes with/without heat treatment, and the OER activities of the resulting films were evaluated. Multiple spectroscopic techniques were performed on the surface of the electrocatalysts to gain insight into the structure-function relationships based on the heterometallic composition. Depending on film preparation, the Co-Mn-oxide was found to change metal composition during catalysis, while the Ni-Mn oxide maintained the NiMn3 ratio. These studies represent the use of discrete heterometallic-oxide clusters as precursors for heterogeneous water oxidation catalysts.
\r\n\r\nAppendix A describes the ongoing effort to synthesize a series of heteromultimetallic [MMn3X] clusters (X = O, S, F). Complexes such as [ZnMn3O], [CoMn3O], [Mn3S], and [Mn4F] have been synthesized and structurally characterized. An amino-bis-oxime ligand (PRABO) has been installed on the [ZnMn3O] cluster. Upon the addition of O2, the desymmetrized [ZnMn3O] cluster only underwent an outer-sphere, one-electron oxidation. Efforts to build and manipulate other heterometallic [MMn3X] clusters are still ongoing, targeting O2 binding and reduction. Appendix B summarizes the multiple synthetic approaches to build a [Co4O4]-cubane complex relevant to heterogeneous OER electrocatalysis. Starting with the tricobalt cluster [LCo3(O2CR)3] and treatment various strong oxidants that can serve as oxygen atom source in the presence Co2+ salt only yielded tricobalt mono\u2013oxo complexes. Appendix C presents the efforts to model the H-cluster framework of [FeFe]-hydrogenase by incorporating a synthetic diiron complex onto a protein-supported or a synthetic ligand-supported [Fe4S4]-cluster. The mutant ferredoxin with a [Fe4S4]-cluster and triscarbene ligand have been characterized by multiple spectroscopic techniques. The reconstruction of an H-cluster mimic has not yet been achieved, due to the difficulty of obtaining crystallographic evidence and the ambiguity of the EPR results.
\r\n", "doi": "10.7907/Z9668B43", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8878", "collection": "thesis", "collection_id": "8878", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212015-194407438", "primary_object_url": { "basename": "Wu_Yunji_2015_thesis.pdf", "content": "final", "filesize": 64667179, "license": "other", "mime_type": "application/pdf", "url": "/8878/1/Wu_Yunji_2015_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein", "author": [ { "family_name": "Wu", "given_name": "Yunji", "clpid": "Wu-Yunji" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Baltimore", "given_name": "David L.", "orcid": "0000-0001-8723-8190", "clpid": "Baltimore-D-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design. ", "doi": "10.7907/Z98050K6", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8537", "collection": "thesis", "collection_id": "8537", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07142014-132743727", "type": "thesis", "title": "Binding Site Structure and Stoichiometry in Serotonin Type 3 Receptors", "author": [ { "family_name": "Miles", "given_name": "Timothy Francis", "clpid": "Miles-Timothy-Francis" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This dissertation primarily describes studies of serotonin type 3 (5-HT3) receptors of the Cys-loop super-family of ligand gated ion channels. The first chapter provides a general introduction to these important proteins and the methods used to interrogate their structure and function. The second chapter details the delineation of a structural unit of the ligand binding site of homomeric 5-HT3A receptors on which the ligands serotonin (5-HT) and m-chlorophenyl biguanide (mCPBG) are reliant for effective receptor activation. Unnatural amino acid mutagenesis results show that the details of each ligand\u2019s interaction with this organizing feature of the binding site differ, providing insights into general principles of receptor activation.
\r\n\r\nThe third chapter describes a study in which florescent protein fusions of the A and B subunits of the heteromeric 5-HT3AB receptor are employed to determine the subunit stoichiometry and order within functional receptors. Strong evidence is found for an A3B2 stoichiometry with A-A-B-A-B order. The fourth chapter investigates the potential for ligand binding across heteromeric binding sites in the 5-HT3AB receptor. Unlike serotonin, mCPBG is found to bind the receptor at heteromeric binding sites. Further mCPBG is capable of allosterically modulating the response of serotonin on the 5-HT3AB receptor from these heteromeric sites.
\r\n\r\nFinally, the fifth chapter describes progress towards the application of unnatural amino acid mutagenesis to an important new class of proteins, transcription factors. Experiments optimizing novel methods for the detection of function are described, using RAR\u03b1 of the nuclear receptor family of transcription factors.
", "doi": "10.7907/Z9T151MC ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8777", "collection": "thesis", "collection_id": "8777", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03092015-135659710", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 19472312, "license": "other", "mime_type": "application/pdf", "url": "/8777/1/Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Ferrous Iron Sensing and Responding in Pseudomonas aeruginosa", "author": [ { "family_name": "Kreamer", "given_name": "Naomi N.", "clpid": "Kreamer-Naomi-N" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Controlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator\u2019s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa\u2019s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future.", "doi": "10.7907/Z9DN4324", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8967", "collection": "thesis", "collection_id": "8967", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-103853129", "primary_object_url": { "basename": "ALFurstThesisCompiled.pdf", "content": "final", "filesize": 25945747, "license": "other", "mime_type": "application/pdf", "url": "/8967/1/ALFurstThesisCompiled.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Devices for Protein Detection", "author": [ { "family_name": "Furst", "given_name": "Ariel Lesa", "orcid": "0000-0001-9583-9703", "clpid": "Furst-Ariel-Lesa" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the \u03c0-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.
\r\n\r\nFirst, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.
\r\n\r\nUsing low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.
\r\n\r\nFor effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 \u03bcL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.
\r\n\r\nWith the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.
\r\n\r\nAs differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.
\r\n", "doi": "10.7907/Z9KH0K88", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8769", "collection": "thesis", "collection_id": "8769", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02122015-130640999", "type": "thesis", "title": "DNA-Mediated Oxidation of Transcription Factor p53", "author": [ { "family_name": "Schaefer", "given_name": "Kathryn Nicole", "orcid": "0000-0003-0908-3191", "clpid": "Schaefer-Kathryn-Nicole" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the \u03c0-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.
\r\n\r\nTo determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]3+, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.
\r\n\r\nThe chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were 13C2D2-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward 13C2D2-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.
\r\n", "doi": "10.7907/Z9BV7DJQ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:9003", "collection": "thesis", "collection_id": "9003", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052015-162319516", "type": "thesis", "title": "Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting", "author": [ { "family_name": "Gristick", "given_name": "Harry Benjamin", "clpid": "Gristick-Harry-Benjamin" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The proper targeting of membrane proteins is essential to the viability of all cells. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET pathway (Guided Entry of TA proteins). In the yeast pathway, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the central targeting factor, the Get3 ATPase. Although binding of Get4/5 to Get3 is critical for efficient TA targeting, the mechanisms by which Get4 regulates Get3 are unknown. To understand the molecular basis of Get4 function, we used a combination of structural biology, biochemistry, and cell biology. Get4/5 binds across the Get3 dimer interface, in an orientation only compatible with a closed Get3, providing insight into the role of nucleotide in complex formation. Additionally, this structure reveals two functionally distinct binding interfaces for anchoring and ATPase regulation, and loss of the regulatory interface leads to strong defects in vitro and in vivo. Additional crystal structures of the Get3-Get4/5 complex give rise to an alternate conformation, which represents an initial binding interaction mediated by electrostatics that facilitates the rate of subsequent inhibited complex formation. This interface is supported by an in-depth kinetic analysis of the Get3-Get4/5 interaction confirming the two-step complex formation. These results allow us to generate a refined model for Get4/5 function in TA targeting.", "doi": "10.7907/Z95718ZD", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8247", "collection": "thesis", "collection_id": "8247", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202014-104709210", "primary_object_url": { "basename": "David Akopian_THESIS.pdf", "content": "final", "filesize": 7517453, "license": "other", "mime_type": "application/pdf", "url": "/8247/1/David Akopian_THESIS.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Phospholipids and a Protein-Conducting Channel Regulate Cotranslational Protein Targeting", "author": [ { "family_name": "Akopian", "given_name": "David", "clpid": "Akopian-David" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Signal recognition particle (SRP) and signal recognition particle receptor (SR) are evolutionarily conserved GTPases that deliver secretory and membrane proteins to the protein-conducting channel Sec61 complex in the lipid bilayer of the endoplasmic reticulum in eukaryotes or the SecYEG complex in the inner membrane of bacteria. Unlike the canonical Ras-type GTPases, SRP and SR are activated via nucleotide-dependent heterodimerization. Upon formation of the SR\u2022SRP targeting complex, SRP and SR undergo a series of discrete conformational changes that culminate in their reciprocal activation and hydrolysis of GTP. How the SR\u2022SRP GTPase cycle is regulated and coupled to the delivery of the cargo protein to the protein-conducting channel at the target membrane is not well-understood. Here we examine the role of the lipid bilayer and SecYEG in regulation of the SRP-mediated protein targeting pathway and show that they serve as important biological cues that spatially control the targeting reaction.
\r\n\r\nIn the first chapter, we show that anionic phospholipids of the inner membrane activate the bacterial SR, FtsY, and favor the late conformational states of the targeting complex conducive to efficient unloading of the cargo. The results of our studies suggest that the lipid bilayer acts as a spatial cue that weakens the interaction of the cargo protein with SRP and primes the complex for unloading its cargo onto SecYEG.
\r\n\r\nIn the second chapter, we focus on the effect of SecYEG on the conformational states and activity of the targeting complex. While phospholipids prime the complex for unloading its cargo, they are insufficient to trigger hydrolysis of GTP and the release of the cargo from the complex. SecYEG modulates the conformation of the targeting complex and triggers the GTP hydrolysis from the complex, thus driving the targeting reaction to completion. The results of this study suggest that SecYEG is not a passive recipient of the cargo protein; rather, it actively releases the cargo from the targeting complex. Together, anionic phospholipids and SecYEG serve distinct yet complementary roles. They spatially control the targeting reaction in a sequential manner, ensuring efficient delivery and unloading of the cargo protein.
\r\n\r\nIn the third chapter, we reconstitute the transfer reaction in vitro and visualize it in real time. We show that the ribosome-nascent chain complex is transferred to SecYEG via a stepwise mechanism with gradual dissolution and formation of the contacts with SRP and SecYEG, respectively, explaining how the cargo is kept tethered to the membrane during the transfer and how its loss to the cytosol is avoided.
\r\n\r\nIn the fourth chapter, we examine interaction of SecYEG with secretory and membrane proteins and attempt to address the role of a novel insertase YidC in this interaction. We show that detergent-solubilized SecYEG is capable of discriminating between the nascent chains of various lengths and engages a signal sequence in a well-defined conformation in the absence of accessory factors. Further, YidC alters the conformation of the signal peptide bound to SecYEG. The results described in this chapter show that YidC affects the SecYEG-nascent chain interaction at early stages of translocation/insertion and suggest a YidC-facilitated mechanism for lateral exit of transmembrane domains from SecYEG into the lipid bilayer.
\r\n\r\n\r\n\r\n\r\n\r\n", "doi": "10.7907/PMBG-Y424", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8027", "collection": "thesis", "collection_id": "8027", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11172013-001549340", "primary_object_url": { "basename": "LeeToniM-2014-Thesis.pdf", "content": "final", "filesize": 27488981, "license": "other", "mime_type": "application/pdf", "url": "/8027/14/LeeToniM-2014-Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Computationally-Guided Thermostabilization of the Primary Endoglucanase from Hypocrea jerorina for Cellulosic Biofuel Production", "author": [ { "family_name": "Lee", "given_name": "Toni Marie", "clpid": "Lee-Toni-Marie" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The creation of thermostable enzymes has wide-ranging applications in industrial, scientific, and pharmaceutical settings. As various stabilization techniques exist, it is often unclear how to best proceed. To this end, we have redesigned Cel5A (HjCel5A) from Hypocrea jecorina (anamorph Trichoderma reesei) to comparatively evaluate several significantly divergent stabilization methods: 1) consensus design, 2) core repacking, 3) helix dipole stabilization, 4) FoldX \u0394\u0394G approximations, 5) Triad \u0394\u0394G approximations, and 6) entropy reduction through backbone stabilization. As several of these techniques require structural data, we initially solved the first crystal structure of HjCel5A to 2.05 \u00c5. Results from the stabilization experiments demonstrate that consensus design works best at accurately predicting highly stabilizing and active mutations. FoldX and helix dipole stabilization, however, also performed well. Both methods rely on structural data and can reveal non-conserved, structure-dependent mutations with high fidelity. HjCel5A is a prime target for stabilization. Capable of cleaving cellulose strands from agricultural waste into fermentable sugars, this protein functions as the primary endoglucanase in an organism commonly used in the sustainable biofuels industry. Creating a long-lived, highly active thermostable HjCel5A would allow cellulose hydrolysis to proceed more efficiently, lowering production expenses. We employed information gleaned during the survey of stabilization techniques to generate HjCel5A variants demonstrating a 12-15 \u00b0C increase in the temperature at which 50% of the total activity persists, an 11-14 \u00b0C increase in optimal operating temperature, and a 60% increase over the maximal amount of hydrolysis achievable using the wild type enzyme. We anticipate that our comparative analysis of stabilization methods will prove useful in future thermostabilization experiments.
", "doi": "10.7907/Z9S180G0", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8044", "collection": "thesis", "collection_id": "8044", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12182013-191308687", "type": "thesis", "title": "Biochemical Studies of Postsynaptic Density Signaling Proteins With a Focus on synGAP and PDZ Domains", "author": [ { "family_name": "Walkup", "given_name": "Ward Gale IV", "orcid": "0000-0002-0385-6256", "clpid": "Walkup-Ward-Gale-IV" } ], "thesis_advisor": [ { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Kennedy", "given_name": "Mary B.", "orcid": "0000-0003-1369-0525", "clpid": "Kennedy-M-B" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Memory storage in the brain involves adjustment of the strength of existing synapses and formation of new neural networks. A key process underlying memory formation is synaptic plasticity, the ability of excitatory synapses to strengthen or weaken their connections in response to patterns of activity between their connected neurons. Synaptic plasticity is governed by the precise pattern of Ca²⁺ influx through postsynaptic N-methyl-D-aspartate-type glutamate receptors (NMDARs), which can lead to the activation of the small GTPases Ras and Rap. Differential activation of Ras and Rap acts to modulate synaptic strength by promoting the insertion or removal of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid receptors (AMPARs) from the synapse. Synaptic GTPase activating protein (synGAP) regulates AMPAR levels by catalyzing the inactivation of GTP-bound (active) Ras or Rap. synGAP is positioned in close proximity to the cytoplasmic tail regions of the NMDAR through its association with the PDZ domains of PSD-95. SynGAP\u2019s activity is regulated by the prominent postsynaptic protein kinase, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5), a known binding partner of CaMKII. Modulation of synGAP\u2019s activity by phosphorylation may alter the ratio of active Ras to Rap in spines, thus pushing the spine towards the insertion or removal of AMPARs, subsequently strengthening or weakening the synapse. To date, all biochemical studies of the regulation of synGAP activity by protein kinases have utilized impure preparations of membrane bound synGAP. Here we have clarified the effects of phosphorylation of synGAP on its Ras and Rap GAP activities by preparing and utilizing purified, soluble recombinant synGAP, Ras, Rap, CaMKII, CDK5, PLK2, and CaM. Using mass spectrometry, we have confirmed the presence of previously identified CaMKII and CDK5 sites in synGAP, and have identified novel sites of phosphorylation by CaMKII, CDK5, and PLK2. We have shown that the net effect of phosphorylation of synGAP by CaMKII, CDK5, and PLK2 is an increase in its GAP activity toward HRas and Rap1. In contrast, there is no effect on its GAP activity toward Rap2. Additionally, by assaying the GAP activity of phosphomimetic synGAP mutants, we have been able to hypothesize the effects of CDK5 phosphorylation at specific sites in synGAP. In the course of this work, we also found, unexpectedly, that synGAP is itself a Ca²⁺/CaM binding protein. While Ca²⁺/CaM binding does not directly affect synGAP activity, it causes a conformational change in synGAP that increases the rate of its phosphorylation and exposes additional phosphorylation sites that are inaccessible in the absence of Ca²⁺/CaM.
\r\n\r\nThe postsynaptic density (PSD) is an electron-dense region in excitatory postsynaptic neurons that contains a high concentration of glutamate receptors, cytoskeletal proteins, and associated signaling enzymes. Within the PSD, three major classes of scaffolding molecules function to organize signaling enzymes and glutamate receptors. PDZ domains present in the Shank and PSD-95 scaffolds families serve to physically link AMPARs and NMDARs to signaling molecules in the PSD. Because of the specificity and high affinity of PDZ domains for their ligands, I reasoned that these interacting pairs could provide the core components of an affinity chromatography system, including affinity resins, affinity tags, and elution agents. I show that affinity columns containing the PDZ domains of PSD-95 can be used to purify active PDZ domain-binding proteins to very high purity in a single step. Five heterologously expressed neuronal proteins containing endogenous PDZ domain ligands (NMDAR GluN2B subunit Tail, synGAP, neuronal nitric oxide synthase PDZ domain, cysteine rich interactor of PDZ three and cypin) were purified using PDZ domain resin, with synthetic peptides having the sequences of cognate PDZ domain ligands used as elution agents. I also show that conjugation of PDZ domain-related affinity tags to Proteins Of Interest (POIs) that do not contain endogenous PDZ domains or ligands does not alter protein activity and enables purification of the POIs on PDZ domain-related affinity resins.
", "doi": "10.7907/Z9N877R8", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8054", "collection": "thesis", "collection_id": "8054", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01182014-130629055", "primary_object_url": { "basename": "Compiled final submission.pdf", "content": "final", "filesize": 30024752, "license": "other", "mime_type": "application/pdf", "url": "/8054/1/Compiled final submission.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Chemical-Scale Studies of G Protein-Coupled Receptors and Ligand-Gated Ion Channels", "author": [ { "family_name": "Van Arnam", "given_name": "Ethan Buggie", "clpid": "Van-Arnam-Ethan-Buggie" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation describes studies of G protein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs) using unnatural amino acid mutagenesis to gain high precision insights into the function of these important membrane proteins.
\r\n \r\nChapter 2 considers the functional role of highly conserved proline residues within the transmembrane helices of the D2 dopamine GPCR. Through mutagenesis employing unnatural \u03b1-hydroxy acids, proline analogs, and N-methyl amino acids, we find that lack of backbone hydrogen bond donor ability is important to proline function. At one proline site we additionally find that a substituent on the proline backbone N is important to receptor function.
\r\n \r\nIn Chapter 3, side chain conformation is probed by mutagenesis of GPCRs and the muscle-type nAChR. Specific side chain rearrangements of highly conserved residues have been proposed to accompany activation of these receptors. These rearrangements were probed using conformationally-biased \u03b2-substituted analogs of Trp and Phe and unnatural stereoisomers of Thr and Ile. We also modeled the conformational bias of the unnatural Trp and Phe analogs employed.
\r\n \r\nChapters 4 and 5 examine details of ligand binding to nAChRs. Chapter 4 describes a study investigating the importance of hydrogen bonds between ligands and the complementary face of muscle-type and \u03b14\u03b24 nAChRs. A hydrogen bond involving the agonist appears to be important for ligand binding in the muscle-type receptor but not the \u03b14\u03b24 receptor.
\r\n \r\nChapter 5 describes a study characterizing the binding of varenicline, an actively prescribed smoking cessation therapeutic, to the \u03b17 nAChR. Additionally, binding interactions to the complementary face of the \u03b17 binding site were examined for a small panel of agonists. We identified side chains important for binding large agonists such as varenicline, but dispensable for binding the small agonist ACh.
\r\n \r\nChapter 6 describes efforts to image nAChRs site-specifically modified with a fluorophore by unnatural amino acid mutagenesis. While progress was hampered by high levels of fluorescent background, improvements to sample preparation and alternative strategies for fluorophore incorporation are described.
\r\n \r\nChapter 7 describes efforts toward a fluorescence assay for G protein association with a GPCR, with the ultimate goal of probing key protein-protein interactions along the G protein/receptor interface. A wide range of fluorescent protein fusions were generated, expressed in Xenopus oocytes, and evaluated for their ability to associate with each other.
\r\n", "doi": "10.7907/0XEB-8013", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8197", "collection": "thesis", "collection_id": "8197", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04222014-120506633", "primary_object_url": { "basename": "Holland_thesis_final.pdf", "content": "final", "filesize": 7943636, "license": "other", "mime_type": "application/pdf", "url": "/8197/1/Holland_thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Design, Construction, and Applications of a High-Resolution Terahertz Time-Domain Spectrometer", "author": [ { "family_name": "Holland", "given_name": "Daniel Brian", "clpid": "Holland-Daniel-Brian" } ], "thesis_advisor": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "local_group": [ { "literal": "Astronomy Department" }, { "literal": "div_chem" } ], "abstract": "This thesis reports on the design, construction, and initial applications of a high-resolution terahertz time-domain ASOPS spectrometer. The instrument employs asynchronous optical sampling (ASOPS) between two Ti:sapphire ultrafast lasers operating at a repetition rate of approximately 80 MHz, and we thus demonstrate a THz frequency resolution approaching the limit of that repetition rate. This is an order of magnitude improvement in resolution over typical THz time-domain spectrometers. The improved resolution is important for our primary effort of collecting THz spectra for far-infrared astronomy. We report on various spectroscopic applications including the THz rotational spectrum of water, where we achieve a mean frequency error, relative to established line centers, of 27.0 MHz. We also demonstrate application of the THz system to the long-duration observation of a coherent magnon mode in a anti-ferromagnetic yttrium iron oxide (YFeO3) crystal. Furthermore, we apply the all-optical virtual delay line of ASOPS to a transient thermoreflectance experiment for quickly measuring the thermal conductivity of semiconductors.", "doi": "10.7907/Z91J97QR", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8197", "collection": "thesis", "collection_id": "8197", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04222014-120506633", "primary_object_url": { "basename": "Holland_thesis_final.pdf", "content": "final", "filesize": 7943636, "license": "other", "mime_type": "application/pdf", "url": "/8197/1/Holland_thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Design, Construction, and Applications of a High-Resolution Terahertz Time-Domain Spectrometer", "author": [ { "family_name": "Holland", "given_name": "Daniel Brian", "clpid": "Holland-Daniel-Brian" } ], "thesis_advisor": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "local_group": [ { "literal": "Astronomy Department" }, { "literal": "div_chem" } ], "abstract": "This thesis reports on the design, construction, and initial applications of a high-resolution terahertz time-domain ASOPS spectrometer. The instrument employs asynchronous optical sampling (ASOPS) between two Ti:sapphire ultrafast lasers operating at a repetition rate of approximately 80 MHz, and we thus demonstrate a THz frequency resolution approaching the limit of that repetition rate. This is an order of magnitude improvement in resolution over typical THz time-domain spectrometers. The improved resolution is important for our primary effort of collecting THz spectra for far-infrared astronomy. We report on various spectroscopic applications including the THz rotational spectrum of water, where we achieve a mean frequency error, relative to established line centers, of 27.0 MHz. We also demonstrate application of the THz system to the long-duration observation of a coherent magnon mode in a anti-ferromagnetic yttrium iron oxide (YFeO3) crystal. Furthermore, we apply the all-optical virtual delay line of ASOPS to a transient thermoreflectance experiment for quickly measuring the thermal conductivity of semiconductors.", "doi": "10.7907/Z91J97QR", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8441", "collection": "thesis", "collection_id": "8441", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302014-103528106", "primary_object_url": { "basename": "thesis_aileen.pdf", "content": "final", "filesize": 8990552, "license": "other", "mime_type": "application/pdf", "url": "/8441/1/thesis_aileen.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanisms of Substrate Selection by the Signal Recognition Particle", "author": [ { "family_name": "Ariosa", "given_name": "Aileen Renia", "clpid": "Ariosa-Aileen-Renia" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The signal recognition particle (SRP) targets membrane and secretory proteins to their correct cellular destination with remarkably high fidelity. Previous studies have shown that multiple checkpoints exist within this targeting pathway that allows \u2018correct cargo\u2019 to be quickly and efficiently targeted and for \u2018incorrect cargo\u2019 to be promptly rejected. In this work, we delved further into understanding the mechanisms of how substrates are selected or discarded by the SRP. First, we discovered the role of the SRP fingerloop and how it activates the SRP and SRP receptor (SR) GTPases to target and unload cargo in response to signal sequence binding. Second, we learned how an \u2018avoidance signal\u2019 found in the bacterial autotransporter, EspP, allows this protein to escape the SRP pathway by causing the SRP and SR to form a \u2018distorted\u2019 complex that is inefficient in delivering the cargo to the membrane. Lastly, we determined how Trigger Factor, a co-translational chaperone, helps SRP discriminate against \u2018incorrect cargo\u2019 at three distinct stages: SRP binding to RNC; targeting of RNC to the membrane via SRP-FtsY assembly; and stronger antagonism of SRP targeting of ribosomes bearing nascent polypeptides that exceed a critical length. Overall, results delineate the rich underlying mechanisms by which SRP recognizes its substrates, which in turn activates the targeting pathway and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved by this protein targeting machinery. ", "doi": "10.7907/AEDK-0925", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8101", "collection": "thesis", "collection_id": "8101", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02282014-131036761", "primary_object_url": { "basename": "ABerry_Feb202014_SubThesis.pdf", "content": "final", "filesize": 12446503, "license": "other", "mime_type": "application/pdf", "url": "/8101/1/ABerry_Feb202014_SubThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Designing Conformational Control of Human Tissue Transglutaminase", "author": [ { "family_name": "Berry", "given_name": "Alexandria Helen T.", "clpid": "Berry-Alexandria-Helen-T" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Understanding the mechanisms of enzymes is crucial for our understanding of their role in biology and for designing methods to perturb or harness their activities for medical treatments, industrial processes, or biological engineering. One aspect of enzymes that makes them difficult to fully understand is that they are in constant motion, and these motions and the conformations adopted throughout these transitions often play a role in their function.
\r\n\r\nTraditionally, it has been difficult to isolate a protein in a particular conformation to determine what role each form plays in the reaction or biology of that enzyme. A new technology, computational protein design, makes the isolation of various conformations possible, and therefore is an extremely powerful tool in enabling a fuller understanding of the role a protein conformation plays in various biological processes.
\r\n\r\nOne such protein that undergoes large structural shifts during different activities is human type II transglutaminase (TG2). TG2 is an enzyme that exists in two dramatically different conformational states: (1) an open, extended form, which is adopted upon the binding of calcium, and (2) a closed, compact form, which is adopted upon the binding of GTP or GDP. TG2 possess two separate active sites, each with a radically different activity. This open, calcium-bound form of TG2 is believed to act as a transglutaminse, where it catalyzes the formation of an isopeptide bond between the sidechain of a peptide-bound glutamine and a primary amine. The closed, GTP-bound conformation is believed to act as a GTPase. TG2 is also implicated in a variety of biological and pathological processes.
\r\n\r\nTo better understand the effects of TG2\u2019s conformations on its activities and pathological processes, we set out to design variants of TG2 isolated in either the closed or open conformations. We were able to design open-locked and closed-biased TG2 variants, and use these designs to unseat the current understanding of the activities and their concurrent conformations of TG2 and explore each conformation\u2019s role in celiac disease models. This work also enabled us to help explain older confusing results in regards to this enzyme and its activities. The new model for TG2 activity has immense implications for our understanding of its functional capabilities in various environments, and for our ability to understand which conformations need to be inhibited in the design of new drugs for diseases in which TG2\u2019s activities are believed to elicit pathological effects.
\r\n", "doi": "10.7907/WW1E-VT19", "publication_date": "2014-06-13", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8212", "collection": "thesis", "collection_id": "8212", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05042014-135648744", "primary_object_url": { "basename": "Daeffler May 2 2014.pdf", "content": "final", "filesize": 21926468, "license": "other", "mime_type": "application/pdf", "url": "/8212/1/Daeffler May 2 2014.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Functional Evaluation of Noncovalent Interactions in Neuroreceptors and Progress Toward the Expansion of Unnatural Amino Acid Methodology", "author": [ { "family_name": "Daeffler", "given_name": "Kristina Nicole-McCleary", "clpid": "Daeffler-Kristina-Nicole-McCleary" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.
\r\n\r\nIn chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-\u03c0 interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-\u03c0 interaction with dopamine.
\r\n\r\nChapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.
\r\n\r\nChapter 4 identifies a cation-\u03c0 interaction between glutamate and a tyrosine residue on loop C in the GluCl\u03b2 receptor. Using the recently published crystal structure of the homologous GluCl\u03b1 receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.
\r\n\r\nChapter 5 examines inherent properties of the GluCl\u03b1 receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.
", "doi": "10.7907/ST7S-DB65", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8225", "collection": "thesis", "collection_id": "8225", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05072014-160207088", "primary_object_url": { "basename": "OC Loson Thesis 2014.pdf", "content": "final", "filesize": 6805019, "license": "other", "mime_type": "application/pdf", "url": "/8225/1/OC Loson Thesis 2014.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Regulation of Mitochondrial Division by the Drp1 Receptors", "author": [ { "family_name": "Loson", "given_name": "Oliver Calvin", "clpid": "Loson-Oliver-Calvin" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Elowitz", "given_name": "Michael B.", "clpid": "Elowitz-M-B" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.
\r\n\r\nThe functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins\u2014Mff, MiD49, and MiD51\u2014play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.
\r\n\r\nWe have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.
\r\n\r\nTo better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.
\r\n", "doi": "10.7907/J23G-KQ18", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8237", "collection": "thesis", "collection_id": "8237", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152014-153300531", "primary_object_url": { "basename": "VowlesJames2014Thesis.pdf", "content": "final", "filesize": 12836579, "license": "other", "mime_type": "application/pdf", "url": "/8237/37/VowlesJames2014Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Cell-Targeted Regulation of Gene Expression through Synthetic RNA Devices", "author": [ { "family_name": "Vowles", "given_name": "James Vincent", "clpid": "Vowles-James-Vincent" } ], "thesis_advisor": [ { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Smolke", "given_name": "Christina D.", "clpid": "Smolke-C-D" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.
\r\n\r\nWe describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.
\r\n\r\nWe next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.
\r\n\r\nThe foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.
", "doi": "10.7907/XE3K-QA07", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8446", "collection": "thesis", "collection_id": "8446", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302014-140200007", "primary_object_url": { "basename": "Su_Judith_2014_thesis.pdf", "content": "final", "filesize": 98921756, "license": "other", "mime_type": "application/pdf", "url": "/8446/1/Su_Judith_2014_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Label-Free Detection of Single Molecule Using Microtoroid Optical Resonators", "author": [ { "family_name": "Su", "given_name": "Tsu-Te Judith", "clpid": "Su-Tsu-Te-Judith" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Vahala", "given_name": "Kerry J.", "orcid": "0000-0003-1783-1380", "clpid": "Vahala-K-J" }, { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_bbe" } ], "abstract": "Being able to detect a single molecule without the use of labels has been a long standing goal of bioengineers and physicists. This would simplify applications ranging from single molecular binding studies to those involving public health and security, improved drug screening, medical diagnostics, and genome sequencing. One promising technique that has the potential to detect single molecules is the microtoroid optical resonator. The main obstacle to detecting single molecules, however, is decreasing the noise level of the measurements such that a single molecule can be distinguished from background. We have used laser frequency locking in combination with balanced detection and data processing techniques to reduce the noise level of these devices and report the detection of a wide range of nanoscale objects ranging from nanoparticles with radii from 100 to 2.5 nm, to exosomes, ribosomes, and single protein molecules (mouse immunoglobulin G and human interleukin-2). We further extend the exosome results towards creating a non-invasive tumor biopsy assay. Our results, covering several orders of magnitude of particle radius (100 nm to 2 nm), agree with the 'reactive' model prediction for the frequency shift of the resonator upon particle binding. In addition, we demonstrate that molecular weight may be estimated from the frequency shift through a simple formula, thus providing a basis for an ``optical mass spectrometer'' in solution. We anticipate that our results will enable many applications, including more sensitive medical diagnostics and fundamental studies of single receptor-ligand and protein-protein interactions in real time. The thesis summarizes what we have achieved thus far and shows that the goal of detecting a single molecule without the use of labels can now be realized.", "doi": "10.7907/EHWP-DH17", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8172", "collection": "thesis", "collection_id": "8172", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03272014-175135389", "type": "thesis", "title": "Electron Flow Through Cytochrome P450", "author": [ { "family_name": "Ener", "given_name": "Maraia Emily", "clpid": "Ener-Maraia-Emily" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The cytochromes P450 (P450s) are a remarkable class of heme enzymes that catalyze the metabolism of xenobiotics and the biosynthesis of signaling molecules. Controlled electron flow into the thiolate-ligated heme active site allows P450s to activate molecular oxygen and hydroxylate aliphatic C\u2013H bonds via the formation of high-valent metal-oxo intermediates (compounds I and II). Due to the reactive nature and short lifetimes of these intermediates, many of the fundamental steps in catalysis have not been observed directly. The Gray group and others have developed photochemical methods, known as \u201cflash-quench,\u201d for triggering electron transfer (ET) and generating redox intermediates in proteins in the absence of native ET partners. Photo-triggering affords a high degree of temporal precision for the gating of an ET event; the initial ET and subsequent reactions can be monitored on the nanosecond-to-second timescale using transient absorption (TA) spectroscopies. Chapter 1 catalogues critical aspects of P450 structure and mechanism, including the native pathway for formation of compound I, and outlines the development of photochemical processes that can be used to artificially trigger ET in proteins. Chapters 2 and 3 describe the development of these photochemical methods to establish electronic communication between a photosensitizer and the buried P450 heme. Chapter 2 describes the design and characterization of a Ru-P450-BM3 conjugate containing a ruthenium photosensitizer covalently tethered to the P450 surface, and nanosecond-to-second kinetics of the photo-triggered ET event are presented. By analyzing data at multiple wavelengths, we have identified the formation of multiple ET intermediates, including the catalytically relevant compound II; this intermediate is generated by oxidation of a bound water molecule in the ferric resting state enzyme. The work in Chapter 3 probes the role of a tryptophan residue situated between the photosensitizer and heme in the aforementioned Ru-P450 BM3 conjugate. Replacement of this tryptophan with histidine does not perturb the P450 structure, yet it completely eliminates the ET reactivity described in Chapter 2. The presence of an analogous tryptophan in Ru-P450 CYP119 conjugates also is necessary for observing oxidative ET, but the yield of heme oxidation is lower. Chapter 4 offers a basic description of the theoretical underpinnings required to analyze ET. Single-step ET theory is first presented, followed by extensions to multistep ET: electron \u201chopping.\u201d The generation of \u201chopping maps\u201d and use of a hopping map program to analyze the rate advantage of hopping over single-step ET is described, beginning with an established rhenium-tryptophan-azurin hopping system. This ET analysis is then applied to the Ru-tryptophan-P450 systems described in Chapter 2; this strongly supports the presence of hopping in Ru-P450 conjugates. Chapter 5 explores the implementation of flash-quench and other phototriggered methods to examine the native reductive ET and gas binding events that activate molecular oxygen. In particular, TA kinetics that demonstrate heme reduction on the microsecond timescale for four Ru-P450 conjugates are presented. In addition, we implement laser flash-photolysis of P450 ferrous\u2013CO to study the rates of CO rebinding in the thermophilic P450 CYP119 at variable temperature. Chapter 6 describes the development and implementation of air-sensitive potentiometric redox titrations to determine the solution reduction potentials of a series of P450 BM3 mutants, which were designed for non-native cyclopropanation of styrene in vivo. An important conclusion from this work is that substitution of the axial cysteine for serine shifts the wild type reduction potential positive by 130 mV, facilitating reduction by biological redox cofactors in the presence of poorly-bound substrates. While this mutation abolishes oxygenation activity, these mutants are capable of catalyzing the cyclopropanation of styrene, even within the confines of an E. coli cell. Four appendices are also provided, including photochemical heme oxidation in ruthenium-modified nitric oxide synthase (Appendix A), general protocols (Appendix B), Chapter-specific notes (Appendix C) and Matlab scripts used for data analysis (Appendix D).", "doi": "10.7907/CFD7-DK06", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8398", "collection": "thesis", "collection_id": "8398", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272014-114201666", "primary_object_url": { "basename": "Deyle_Kaycie_2014_Thesis_Complete.pdf", "content": "final", "filesize": 3692951, "license": "other", "mime_type": "application/pdf", "url": "/8398/1/Deyle_Kaycie_2014_Thesis_Complete.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Development of Protein-Catalyzed Capture (PCC) Agents with Application to the Specific Targeting of the E17K Point Mutation of AKt1", "author": [ { "family_name": "Deyle", "given_name": "Kaycie Marie", "clpid": "Deyle-Kaycie-Marie" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.
\r\n\r\nThese technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.
", "doi": "10.7907/F8HW-TX51", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8412", "collection": "thesis", "collection_id": "8412", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282014-143107089", "primary_object_url": { "basename": "thesis_final.pdf", "content": "final", "filesize": 8258010, "license": "other", "mime_type": "application/pdf", "url": "/8412/1/thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Design, Synthesis, and Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Komor", "given_name": "Alexis Christine", "clpid": "Komor-Alexis-Christine" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.
\r\n \r\nHere we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.
\r\n\r\nIn addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.
\r\n\r\nTo explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.
\r\n\r\nFinally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both \u0394 and \u039b enantiomers of [Rh(chrysi)(phen)(DPE)]2+ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.
\r\n", "doi": "10.7907/KPCY-JS09", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8415", "collection": "thesis", "collection_id": "8415", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282014-200718606", "primary_object_url": { "basename": "Crystal N Dilworth Thesis Final Upload.pdf", "content": "final", "filesize": 8253852, "license": "other", "mime_type": "application/pdf", "url": "/8415/25/Crystal N Dilworth Thesis Final Upload.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Fluorescence Microscopy of Nicotinic Acetylcholine Receptors", "author": [ { "family_name": "Dilworth", "given_name": "Crystal Noelle", "clpid": "Dilworth-Crystal-Noelle" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels abundantly expressed in the central nervous system. Changes in the assembly and trafficking of nAChRs are pertinent to disease states including nicotine dependence, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), and Parkinson\u2019s disease (PD). Here we investigate the application of high resolution fluorescence techniques for the study of nAChR assembly and trafficking. We also describe the construction and validation of a fluorescent \u03b15 subunit and subsequent experiments to elucidate the cellular mechanisms through which \u03b15 subunits are expressed, assembled into mature receptors, and trafficked to the cell surface. The effects of a known single nucleotide polymorphism (D398N) in the intracellular loop of \u03b15 are also examined.
\r\n\r\nAdditionally, this report describes the development of a combined total internal reflection fluorescence (TIRF) and lifetime imaging (FLIM) technique and the first application of this methodology for elucidation of stochiometric composition of nAChRs. Many distinct subunit combinations can form functional receptors. Receptor composition and stoichiometry confers unique biophysical and pharmacological properties to each receptor sub-type. Understanding the nature of assembly and expression of each receptor subtype yields important information about the molecular processes that may underlie the mechanisms through which nAChR contribute to disease and addiction states.
", "doi": "10.7907/Z96D5QZB", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7204", "collection": "thesis", "collection_id": "7204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09132012-234333515", "primary_object_url": { "basename": "dropbox.cgi_get=1&key=6e0eff88ef58320721332530c754ba2c", "content": "final", "filesize": 8549662, "license": "other", "mime_type": "application/pdf", "url": "/7204/1/dropbox.cgi_get=1&key=6e0eff88ef58320721332530c754ba2c", "version": "v4.0.0" }, "type": "thesis", "title": "Applications of Computational Protein Design to Red Fluorescent Proteins", "author": [ { "family_name": "Moore", "given_name": "Matthew Michaels", "clpid": "Moore-Matthew-Michaels" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The research comprising this thesis is presented in three chapters divided into two parts. Part One, Chapters 2 and 3, of the thesis focuses on the application of rational design and CPD to the core residues of FPs. Chapter 2 applies CPD to the well known red fluorescent protein mCherry. Design hypotheses in this work were driven by a desire to red-shift the fluorescence emission of the parent protein. Chapter 3 takes the most successful results from the mCherry system and attempts to applies these results to the far-red FP mPlum. These two proteins, mCherry and mPlum, share a directed evolution parent, mRFP1. Part Two, consisting of Chapter 4, presents the beginnings of a comprehensive study into the applications CPD for designing FP surfaces. The system used is based on DsRed, the oligomeric parent of monomer mCherry. Professional and personal acknowledgments conclude the thesis.", "doi": "10.7907/6876-A570", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7710", "collection": "thesis", "collection_id": "7710", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142013-213725125", "type": "thesis", "title": "Single-Cell Analysis of the Physiology of Mechanosensation in Bacteria", "author": [ { "family_name": "Bialecka-Fornal", "given_name": "Maja I.", "clpid": "Bialecka-Fornal-Maja-I" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Escherichia coli is one of the best studied living organisms and a model system for many biophysical investigations. Despite countless discoveries of the details of its physiology, we still lack a holistic understanding of how these bacteria react to changes in their environment. One of the most important examples is their response to osmotic shock. One of the mechanistic elements protecting cell integrity upon exposure to sudden changes of osmolarity is the presence of mechanosensitive channels in the cell membrane. These channels are believed to act as tension release valves protecting the inner membrane from rupturing. This thesis presents an experimental study of various aspects of mechanosensation in bacteria. We examine cell survival after osmotic shock and how the number of MscL (Mechanosensitive channel of Large conductance) channels expressed in a cell influences its physiology. We developed an assay that allows real-time monitoring of the rate of the osmotic challenge and direct observation of cell morphology during and after the exposure to osmolarity change. The work described in this thesis introduces tools that can be used to quantitatively determine at the single-cell level the number of expressed proteins (in this case MscL channels) as a function of, e.g., growth conditions. The improvement in our quantitative description of mechanosensation in bacteria allows us to address many, so far unsolved, problems, like the minimal number of channels needed for survival, and can begin to paint a clearer picture of why there are so many distinct types of mechanosensitive channels.", "doi": "10.7907/7SRD-WS94", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7255", "collection": "thesis", "collection_id": "7255", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11052012-192506907", "primary_object_url": { "basename": "wlimapichat_complete_thesis.pdf", "content": "final", "filesize": 7469835, "license": "other", "mime_type": "application/pdf", "url": "/7255/1/wlimapichat_complete_thesis.pdf", "version": "v11.0.0" }, "type": "thesis", "title": "Probing the Roles of Receptor Structure, Drug-Receptor Interactions, and Receptor Crosstalk in Ligand-Gated Ion Channel Function", "author": [ { "family_name": "Limapichat", "given_name": "Walrati", "clpid": "Limapichat-Walrati" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ligand-gated ion channels are multi-subunit transmembrane proteins that play crucial roles in synaptic transmission in the nervous system. These include the Cys-loop receptor superfamily, the ionotropic glutamate receptor (iGluR) family, and the purinergic P2X receptor family. Binding of specific neurotransmitters at the ligand-binding site triggers a series of conformational changes that ultimately leads to ion channel opening. This dissertation describes three molecular-scale functional studies on these receptors.
\r\n\r\nThe first project (Chapter 2) describes structure-function studies of the conserved Phe-Pro motif in the Cys loop of the nicotinic acetylcholine receptor (nAChR) of the Cys-loop superfamily. Both residues were substituted with natural and unnatural amino acids. A strong interaction between the Phe and Pro residues is evident, as is a preference for aromaticity at the Phe site. Hydrophobicity is preferred at both sites. A correlation between receptor function and the cis bias at the proline backbone suggests a significant role for the cis proline conformer in receptor function.
\r\n\r\nThe second project (Chapter 3) concerns the key binding interaction of memantine, a prescribed drug for Alzheimer\u2019s disease, on the N-methyl-D-aspartate (NMDA) receptor of the iGluR family. The data suggest that the special property of memantine as an NMDA receptor blocker stems from the presence of the two methyl groups and a proper shape-matching to the binding site. Comparing affinities of memantine and amantadine, a structurally related drug, in response to pore mutations allows an identification of the methyl group binding pockets on the NMDA channel pore.
\r\n\r\nThe final project (Chapter 4) involves a study of inhibitory crosstalk between two families of ion channels: \u03b16\u03b24-containing nAChRs and P2X receptors. When these two distinct receptors are co-expressed, their properties are modulated from their normal behavior when expressed alone. The effect is constitutive and does not require channel activation. When they are co-activated by their respective agonists, the observed current is smaller than the sum of the currents evoked by individual application of their agonists. This functional interaction between these nicotinic and purinergic receptors in dorsal root ganglion neurons is proposed to be involved in pain sensation.
\r\n", "doi": "10.7907/YPMW-9E79", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7496", "collection": "thesis", "collection_id": "7496", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02282013-171348639", "primary_object_url": { "basename": "BenLi Thesis Final Edit.pdf", "content": "final", "filesize": 10711126, "license": "other", "mime_type": "application/pdf", "url": "/7496/1/BenLi Thesis Final Edit.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Synthesis and Biological Studies of DNA-binding Cyclic Py-Im Polyamides", "author": [ { "family_name": "Li", "given_name": "Benjamin Chun Yeung", "clpid": "Li-Benjamin-Chun-Yeung" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Pyrrole-imidazole (Py-Im) polyamides are programmable oligomers that bind to the minor groove of DNA in a sequence-specific manner at affinities comparable to natural DNA-binding proteins. Hairpin polyamides have been shown to localize within the nucleus of live cells, disrupt protein-DNA interactions, and modulate endogenous gene expression. Cyclic polyamides display further enhanced DNA binding affinities and exhibit similar gene regulatory effects, but investigations into their biological activity have been limited by the lack of effective synthetic methods. Herein, we demonstrate the efficient synthesis of a focused library of cyclic polyamide utilizing a novel microwave-assisted solid-phase technique. The orthogonal protection strategy allowed for selective turn modifications, and the mild cleavage conditions gave access to polyamide cores beginning with a C-terminal imidazole. In addition to expanding our synthetic repertoire, we further examined the cytotoxicity and cell uptake profiles of the cyclic polyamide variants, which highlighted the significant changes in biological activity resulting from minor structural modifications. Molecular recognition of the polyamide turn unit was also explored by installing heteroatom substituents at the \u03b1-position. Interestingly, while none of the fluoro, hydroxyl, or amino derivatives increased turn specificity, the (S)-fluoro turn exhibited better tolerance for binding a C\u2022G pair. Finally, we optimized the synthesis of several biologically active hairpin polyamides on a 50-mg scale and examined their antitumor activity in mice xenograft models.", "doi": "10.7907/82YX-QB29", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7446", "collection": "thesis", "collection_id": "7446", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01282013-083506263", "primary_object_url": { "basename": "Muren_Natalie_2013_combined_chapters.pdf", "content": "final", "filesize": 5695199, "license": "other", "mime_type": "application/pdf", "url": "/7446/1/Muren_Natalie_2013_combined_chapters.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection", "author": [ { "family_name": "Muren", "given_name": "Natalie Bloom", "clpid": "Muren-Natalie-Bloom" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The structural core of DNA, a continuous stack of aromatic heterocycles\u2014the base pairs\u2014that extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. This \u03c0-stacked structure facilitates a unique form of charge conduction, termed DNA-mediated charge transport (DNA CT). Experiments with diverse platforms, in solution, on surfaces, and with single molecules, collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. Notably, DNA CT can proceed over long molecular distances, but is remarkably sensitive to perturbations in base pair stacking. These characteristics suggest that DNA CT may be used for long-range sensing both in nature and in nanoelectronic applications. Here, measurements of DNA CT with surface and single molecule platforms are used to (i) determine how ground state DNA CT varies over regimes of increasing distance and (ii) apply this chemistry to the electrical detection of DNA-binding proteins.
\r\n\r\nFirst, the design and fabrication of multiplexed, DNA-modified electrodes on silicon chips is reported. These lithographically patterned chips with 16 individually addressable gold electrodes allow for the measurement of DNA CT with four different types of DNA, side by side on the same surface, with four-fold redundancy. Discrimination of DNA with a single base mismatch and detection of sequence-specific restriction enzyme activity are both achieved with these chips. Scaling of these devices to microelectrode dimensions is also demonstrated. Importantly, these chips show greater reproducibility and consistency than commercially available rod electrodes. This greater signal quality, combined with the capacity to examine different samples side by side, opens the door for more complex applications of this platform.
\r\n\r\nThe fully developed, multiplexed chips are first used to compare DNA CT over short and long distance regimes. DNA is evaluated in this context because the efficacy of a long-range sensor, in either nature or nanoelectronics, is determined largely by its capacity to facilitate CT in a manner that is minimally affected by the CT distance. DNA CT over 34 nm in 100-mer monolayers is found to yield electrochemical signals that are comparable in size to shorter 17-mer DNA. Signal attenuation from a single base-pair mismatch in the 100-mer is also comparable to that for 17-mers, and confirms that CT in these 100-mer films is DNA-mediated. Efficient cleavage by a restriction enzyme indicates that the 100-mer DNA adopts a native, upright conformation. The alkanethiol linker used to anchor the DNA to the electrode is found to limit the electron-transfer rate for both DNA lengths. Thus the impact of increasing the CT distance on DNA CT is too small to be resolved by this platform, even over 34 nm. These measurements put DNA among the longest and most conductive molecular wires reported to date.
\r\n\r\nNext, DNA CT with multiplexed chips is extended to the electrochemical detection of methyltransferases, proteins that are attractive targets because of their prominent role in the initial stages of many types of cancer. Electrochemical detection of binding and activity by these proteins is achieved by two different methods. First, DNA-binding and base-flipping by these proteins disrupts the DNA \u03c0-stack and may be used for direct \u201csignal OFF\u201d detection. Using this method, the concentration- and cofactor- dependence of SssI methyltransferase, the bacterial analog of human methyltransferases, are examined. Second, methylation-conferred protection of DNA against cutting by a restriction enzyme may be used for \u201csignal ON\u201d detection of methyltransferase activity. With this approach, the use of both unmethylated and hemimethylated DNA substrates is demonstrated for the sensitive detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Importantly, the electrochemical format of these assays requires minimal equipment, is low cost, and may be easily applied to high throughput studies, making it an accessible option for a variety of research and clinical settings.
\r\n\r\nAlongside work with this surface, electrochemical platform, a single molecule, carbon nanotube-DNA (CNT-DNA) platform is also used to evaluate DNA CT over increasing distances and to detect protein binding. CNT-DNA devices consist of a single molecule of DNA that is made to bridge a gap cut in a CNT covalently, such that current flow through the device is DNA-mediated. Upon introduction of DNA bridges of varying length (15-mer, 60-mer, and 100-mer), the device resistance is minimally affected, echoing the result of long distance electrochemistry experiments. These devices are also used to detect SssI methyltransferase binding by the direct \u201csignal OFF\u201d method used with multiplexed chips; DNA-binding and base-flipping disrupts DNA CT and shuts off current flow through the device. CNT-DNA devices are used to electronically measure the sequence-specific, cofactor-dependent, and reversible binding of SssI. DNA methylation catalyzed by SssI is also detected based on its alteration of the protein-binding affinity of the device. This detection approach, which relies on DNA as both a recognition element and electrical transducer, represents a unique strategy for the specific, single molecule detection of protein binding and activity.
\r\n", "doi": "10.7907/6KG5-KQ87", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7444", "collection": "thesis", "collection_id": "7444", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01272013-150337805", "primary_object_url": { "basename": "medium.jpg", "content": "", "filesize": 6725, "license": "other", "mime_type": "image/png", "url": "/7444/1/medium.jpg", "version": "v2.0.0" }, "type": "thesis", "title": "Discovery of New Roles for Chondroitin Sulfate in Neurotrophin Signaling and Retinotopic Development", "author": [ { "family_name": "Rogers", "given_name": "Claude Joseph", "clpid": "Rogers-Claude-Joseph" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chondroitin sulfate (CS), a member of the glycosaminoglycan family of linear polysaccharides, is involved in the formation and maintenance of neuronal networks. CS has dual roles in regulating neuronal morphology: promoting or inhibiting neuronal outgrowth, depending on the context. A single sulfated epitope, CS-E, is capable of inducing both types of activity.
\r\n\r\nMembers of the neurotrophin (NT) family of growth factors are required for CS- E-induced neurite outgrowth in hippocampal neurons. Here, we demonstrate that CS is capable of forming ternary complexes with NTs and their receptors. These complexes were discovered using a novel, carbohydrate microarray-based approach that allows for the rapid screening of such interactions. To support these findings, we computationally determined the CS-E-binding site of the complexes, suggesting a structural basis for the interaction. In addition, we showed that CS-E is capable of attenuating NT signaling in cells, consistent with our computational and microarray data. This is the first demonstration that CS-E is involved in NT signaling and that CS is capable of supporting multimeric signaling complexes.
\r\n\r\nIn addition to stimulating growth factor signaling, CS has been known to repulsively guide retinal ganglion cell (RGC) axons for over twenty years. However, its function in vivo is unknown. RGCs are the only neuron type that transmits visual information to the brain, and their guidance, which maps a topographic projection of the retina to the superior colliculus (SC), is tightly regulated. Here, we show that CS-E is required for the proper formation of this topographic order. CS-E, but not the other major sulfation patterns, is a repellent guidance cue for RGC axons, with a graded activity profile from low to high along the dorsal-ventral axis of the retina, congruent with EphB3 expression. EphB3 binds specifically to CS-E with physiologically relevant affinity, and is required for CS-E-mediated guidance. CS-E-null mice have defects in topographic mapping in which ventral axons form ectopic termina- tions medial to their correct location in the SC. These results indicate that CS is a repulsive guidance cue required to map the dorsal-ventral axis of the retina along the lateral-medial axis of the SC. This is the first report of a non-protein topographical\r\nguidance cue.
", "doi": "10.7907/Z9N58JCD", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7218", "collection": "thesis", "collection_id": "7218", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10012012-155636249", "primary_object_url": { "basename": "chartron_justin_2013_thesis.pdf", "content": "final", "filesize": 44887356, "license": "other", "mime_type": "application/pdf", "url": "/7218/1/chartron_justin_2013_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Structure of a Transmembrane Protein Sorting Complex", "author": [ { "family_name": "Chartron", "given_name": "Justin William", "clpid": "Chartron-Justin-William" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The biogenesis of membrane proteins is an essential process in biology. It requires the protection of hydrophobic transmembrane domains from aggregation in the cytosol as well as targeting to the proper membrane. Tail-anchored (TA) proteins have a single transmembrane helix near their carboxyl termini and require a post-translational mechanism for targeting and insertion. In yeast, the Guided Entry of Tail-anchored proteins (GET) pathway delivers TA proteins to the endoplasmic reticulum (ER). A sorting complex comprising Get4, Get5, and Sgt2 load ER destined TA proteins onto the targeting factor Get3. X-ray crystallography, solution NMR, and small angle X-ray scattering were used to characterize this assembly. Get4 and Get5 form an extended adapter complex. Get4 maintains Get3 in a state competent to receive TA proteins. The N-terminus of Get5 tightly binds Get4, while the C-terminus of Get5 is a homodimerization domain, resulting in a heterotetrameric assembly. A ubiquitin-like domain within Get5 binds the heat-shock protein (HSP) co-chaperone Sgt2, providing a physical link between ER destined TA protein targeting and protein folding pathways. Sgt2 is also an extended homodimeric complex, and can directly bind four major classes of HSPs. The Get4/Get5/Sgt2 sorting complex is multivalent, flexible and the binding of individual components is transient. These results build a model for post-translational protein targeting in eukaryotes that is distinct from other pathways.", "doi": "10.7907/QVCV-8A76", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7193", "collection": "thesis", "collection_id": "7193", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08282012-110955629", "type": "thesis", "title": "A Novel Method for Studying Nucleated Pathways in Membranes: Development and Applications for Gene Delivery", "author": [ { "family_name": "Ting", "given_name": "Christina Lei", "clpid": "Ting-Christina-Lei" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The development of a safe, selective, and efficient gene delivery system is key to the success of human gene therapy. In polymer-based gene delivery systems, biocompatible polymers electrostatically bind and condense the genetic material into protective nanoparticles. These nanoparticles must subsequently overcome several challenges, which remain poorly understood. In particular, once internalized by the cell, the nanoparticles are trapped inside a membrane-bound compartment called the endosome. In the proton sponge hypothesis, the buffering capacity of the polymers leads to an increase in osmotic pressure that eventually ruptures the endosomal membrane and releases the trapped nanoparticles.
\r\n\r\nTo obtain a mechanistic understanding of the endosomal escape, we first develop a coarse-grained model to study the equilibrium interaction between a positively charged nanoparticle and a lipid membrane. Results indicate the existence of a pore with an inserted particle, whose metastability depends on the membrane tension and particle properties (size and charge). These pores are subsequently shown to lower the critical tension necessary for membrane rupture, thus possibly enhancing the release of the trapped genetic material from the endosome.
\r\n\r\nNext, we address the actual escape pathway, which is likely a thermally nucleated process and cannot be simulated directly or studied by equilibrium methods. Hence, we develop a novel method for studying minimum free energy paths in membranes. Our results indicate that thermally nucleated rupture may be an important factor for the low rupture strains observed in lipid membranes. Under the moderate tensions found in this regime, there are multiple pathways for crossing the membrane: (1) particle-assisted membrane rupture, (2) particle insertion into a metastable pore followed by translocation and membrane resealing, and (3) particle insertion into a metastable pore followed by membrane rupture. This suggests a direct role of the nanoparticle in the endosomal escape not previously envisioned in the proton sponge hypothesis, and illustrates the importance of having an induced tension on the membrane.
\r\n\r\nFinally, the methodology developed in this work represents the most advanced theoretical technique for describing nucleation pathways in soft condensed matter systems that also include hard-particle degrees of freedom. We expect the method to be useful for studying a wide range of nucleation phenomena beyond membrane systems, for example, in nanoparticle polymer composites.
\r\n", "doi": "10.7907/8FTH-8H35", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7775", "collection": "thesis", "collection_id": "7775", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302013-155904165", "primary_object_url": { "basename": "FordNicole2013thesis.pdf", "content": "final", "filesize": 5837787, "license": "other", "mime_type": "application/pdf", "url": "/7775/1/FordNicole2013thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Capturing Protein Dynamics with Time-Resolved Luminescence Spectroscopy", "author": [ { "family_name": "Ford", "given_name": "Nicole Danielle Bouley", "clpid": "Ford-Nicole-Danielle-Bouley" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome
We have also investigated intrachain contact dynamics in unfolded cytochrome
In addition, we have explored the pathway dependence of electron tunneling rates between metal sites in proteins. Our research group has converted cytochrome
The global transcription factor p53 controls many cellular processes, including the cellular response to oxidative stress. It had been determined that that dissociation of wild type p53 from its promoter site can occur upon DNA-mediated oxidation. In this work, we use site-directed mutagenesis to construct charge-deficient mutants of p53; the chemistry of DNA-mediated oxidation of p53 was examined using these mutants.
\r\n\r\nThe control point for p53 oxidation through DNA-mediated charge transport (DNA CT) is cysteine 275. Using differential thiol labeling and detection of modified peptides with mass spectrometry, we demonstrated that cysteines 124 and 141 in superstable p53 form a terminal disulfide bond upon DNA-mediated oxidation. This leads to a conformational change that inhibits DNA from binding by p53. The disulfide formed between cysteines 124 and 141 is a result of a series of disulfide bond exchange across the protein from the DNA base stack.
\r\n\r\nWe also investigated the dependence of p53 oxidation on DNA sequences. ESMA analysis of biologically derived p53 recognition sequences with varying quantities of guanine doublets and triplets showed efficient p53 oxidation to depend on the presence of low energy GG or GGG sites. Moreover, consistent results were found with biologically derived promoter sequences. Sequence S100A2, with guanine triplets on the same strand, showed the most oxidation of p53, followed by ODC1 and caspase-1. We confirmed these sequence-specific effects by measuring the change in expression level of the genes after induction of DNA CT in vivo. S100A2 mRNA levels decreased after photooxidant and light treatment, reflecting the oxidation and dissociation of p53 from the S100A2 site. However, caspase-1 and ODC1 mRNA levels remained the same, indicating less DNA-mediated p53 oxidation.
\r\n\r\nThe results from this study illustrate how protein oxidation at a distance through DNA CT contributes to cellular signaling. This oxidative signaling can control how p53 regulates gene expression under oxidative stress, and this signaling may be disrupted in cancerous cells.
\r\n", "doi": "10.7907/0NVZ-QC23", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6721", "collection": "thesis", "collection_id": "6721", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10282011-143721555", "primary_object_url": { "basename": "Olmon2012thesis.pdf", "content": "final", "filesize": 6459459, "license": "other", "mime_type": "application/pdf", "url": "/6721/1/Olmon2012thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Investigating DNA-Mediated Charge Transport by Time-Resolved Spectroscopy", "author": [ { "family_name": "Olmon", "given_name": "Eric Daniel", "clpid": "Olmon-Eric-Daniel" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In all organisms, oxidation threatens the integrity of the genome. Numerous studies have suggested that DNA-mediated charge transport (CT) may play an important role in the sequestration, detection, and repair of oxidative damage. To fully understand the mechanism of DNA-mediated CT, it is necessary to characterize transient intermediates that arise during the reaction and to determine the lifetimes of these intermediates. Time-resolved spectroscopy is the most appropriate experimental method for such observations. Each intermediate has a characteristic spectrum. By observing time-dependent changes in the absorption spectrum of the sample, it is therefore possible to determine what species are present at a particular time and how long it exists in solution. Experiments presented here involve the use of time-resolved spectroscopy to better understand the process of DNA-mediated CT.
\r\n\r\nThe study of DNA-mediated CT requires a robust and consistent method for triggering the CT reaction. The metal complexes that have traditionally been used for this purpose provide several advantages over organic phototriggers: they are synthetically versatile, they are stable in solution, they exhibit rich photophysics, and many are strong photooxidants. However, the spectroscopic features used to follow the photochemical processes triggered by these probes are generally broad optical bands. These can be difficult to resolve in samples that contain several absorbing species. For this reason, we have developed a Re photooxidant bearing a set of vibrationally active carbonyl ligands that can be covalently tethered to DNA. Unlike many absorption bands in the visible range, the vibrational absorption bands of these ligands are narrow, well-resolved, and specific. Such probes can be used to follow the complex photophysical pathways observed in biochemical systems with good precision, making them useful for the study of DNA-mediated CT.
\r\n\r\nSpecifically, the complex [Re(CO)3(dppz)(py′-OR)]+ (dppz = dipyrido[3,2-a:2′,3′-c]-phenazine; py′-OR = 4-functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state. The behavior of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR (TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the ability of the complex to trigger long-range oxidation of DNA. Optical excitation of the complex leads to population of metal-to-ligand charge transfer excited states and at least two distinct intraligand charge transfer excited states. Several experimental observations are consistent with charge injection by excited Re*. These include similarity between TRIR spectra and the spectrum of reduced Re observed by spectroelectrochemistry, the appearance of a guanine radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation products. The majority of reactivity occurs on the ultrafast time scale, although processes dependent on slower conformational motions of DNA, such as the accumulation of oxidative damage at guanine, are also observed.
\r\n\r\nThe photooxidation activity of this Re complex was compared directly to that of other metallointercalators that have been used previously in our laboratory to oxidize DNA. The complexes [Rh(phi)2(bpy′)]3+ (phi = 9,10-phenanthrenequinone diimine; bpy′ = 4-methyl-4′-(butyric acid)-2,2′-bipyridine), [Ir(ppy)2(dppz′)]+ (ppy = 2-phenylpyridine; dppz′ = 6-(dipyrido[3,2-a:2′,3′-c]phenazin-11-yl)hex-5-ynoic acid), and [Re(CO)3(dppz)(py′-OH)]+ (py′-OH = 3-(pyridin-4-yl)-propanoic acid) were each covalently tethered to DNA. Biochemical studies show that upon irradiation, the three complexes oxidize guanine by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those obtained by transient absorption (TA) spectroscopy of the conjugates suggests that excitation of the conjugates at 355 nm results in the formation of the reduced metal states. Electrochemical experiments and kinetic analysis of the TA decays verify that the primary factors responsible for the trend observed in the guanine oxidation yield of the three complexes are the thermodynamic driving force for CT, variations in the efficiency of back electron transfer, and coupling to DNA.
\r\n\r\nThe ability of redox-active DNA-binding proteins to act as hole sinks in DNA-mediated CT systems was also studied by time-resolved spectroscopy. Such experiments are designed to provide support for the utilization of DNA-mediated CT in biological systems. In studies involving the cell cycle regulator p53, photoexcitation results in the formation of a weak transient band at 405 nm. This band, which is not observed in samples lacking the protein, resembles the primary spectral feature of the tyrosine cation radical. Although the signal is weak and reproducibility is inconsistent, these results suggest that photolysis of the sample leads to DNA-mediated oxidation of tyrosine in p53. Similar experiments were conducted on the transcriptional activator SoxR. Here, the presence of dithionite, required in solution to keep the protein reduced, complicates the photochemistry of the system considerably. Regardless, a weak absorbance at 418 nm that develops following photolysis at 355 nm provides evidence for the DNA-mediated oxidation of the protein. The behavior of the base excision repair protein endonuclease III was also observed in the presence of DNA and metal complex oxidants. In flash-quench studies, addition of the protein results in the formation of a strong negative signal at 410 nm in TA traces. In studies involving direct photooxidation by Rh, Ir, and Re complexes, no new transients are detected upon the addition of protein, but changes in the intensities of the resultant TA spectra and in the steady-state absorbance spectra following photolysis indicate that DNA-mediated oxidation of the protein may be taking place.
\r\n\r\nThe experiments described here comprise several new developments in the story of DNA-mediated CT. First, proof of concept has been given for a valuable new vibrationally-active Re probe. Further modifications on the characteristics of this complex and further study by time-resolved vibrational spectroscopy will allow us to observe DNA-mediated CT with high spectral resolution. Second, comparison between this Re probe and established photooxidants shows that the Re complex is a strong photooxidant in its own right and that this complex can be added to our growing toolbox of CT phototriggers. Third, time-resolved studies involving redox-active proteins have provided preliminary direct evidence for the ability of these proteins to serve as CT probes themselves. Further refinement of the experimental methods used in these experiments will allow us to observe such processes with greater sensitivity, increasing our knowledge of the mechanism and applications of DNA-mediated CT.
\r\n", "doi": "10.7907/6GDE-2707", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6731", "collection": "thesis", "collection_id": "6731", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11072011-185116656", "primary_object_url": { "basename": "Song_thesis_2012.pdf", "content": "final", "filesize": 21244626, "license": "other", "mime_type": "application/pdf", "url": "/6731/1/Song_thesis_2012.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Recognition of Nucleic Acid Mismatches by Luminescent Ruthenium Complexes", "author": [ { "family_name": "Song", "given_name": "Hang", "clpid": "Song-Hang" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Deficiencies in DNA mismatch repair (MMR) have been implicated in the development of several forms of cancers, and MMR-deficient cells tend to be resistant to commonly employed cancer therapeutics such as cisplatin. Mismatch-targeting metalloinsertors developed in our laboratory have shown great promise as therapeutic and diagnostic agents for MMR-deficient cancers. In this work, we examine fundamental aspects of binding interactions of octahedral rhodium and ruthenium complexes to DNA mismatches, and strive to develop a luminescent sensor for mismatches inside cells.
\r\n\r\nWe first demonstrate that the mismatch binding affinity of rhodium metalloinsertors directly correlates with their antiproliferative effect against MMR-deficient colorectal carcinoma cells. Smaller ancillary ligands on the rhodium center facilitate binding to mismatches via metalloinsertion from the narrow minor groove of DNA. Complexes with higher mismatch binding affinity in turn selectively inhibit the growth of MMR-deficient cells compared to MMR-proficient ones. This correlation suggests that DNA mismatches are indeed the biological target of rhodium metalloinsertors inside cells.
\r\n\r\nBesides rhodium metalloinsertors, luminescent ruthenium complexes are found to bind DNA mismatches as well. Mismatch binding is accompanied by enhanced luminescence intensity. We determined two crystal structures of \u0394-Ru(bpy)2dppz2+ bound to oligonucleotide duplexes. For an oligonucleotide containing AA mismatches, the atomic-resolution structure revealed that the ruthenium complex binds to DNA mismatches also through metalloinsertion: the complex inserts a planar ligand into the mismatched site from the minor groove, ejecting the mismatched bases out of the helix. Several binding geometries of the complex intercalated between well-matched DNA were also observed.
\r\n\r\nTo improve the mismatch selectivity of luminescent ruthenium complexes, we tethered the complexes to organic dye molecules in an effort to amplify mismatch-associated luminescence signal through resonance energy transfer. We also modified the structure of the inserting ligand in an attempt to improve the binding affinity to mismatches over well-matched DNA. Coupling mismatch binding to luminescence response has proved most challenging in these endeavors.
\r\n\r\nFinally, we venture into the realm of RNA. Unlike their nonspecific binding to DNA, ruthenium complexes bind poorly to well-matched RNA but quite avidly to RNA mismatches. As a result, mismatched RNA produces a higher luminescence signal from bound ruthenium. We subsequently applied the ruthenium complex to image RNA mismatches inside live HeLa cells using fluorescence microscopy.
", "doi": "10.7907/4TM6-BR89", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6812", "collection": "thesis", "collection_id": "6812", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02062012-154120957", "primary_object_url": { "basename": "Nisthal2012_thesis.pdf", "content": "final", "filesize": 45273265, "license": "other", "mime_type": "application/pdf", "url": "/6812/35/Nisthal2012_thesis.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Accelerating the Interplay Between Theory and Experiment in Protein Design", "author": [ { "family_name": "Nisthal", "given_name": "Alex", "clpid": "Nisthal-Alex" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Protein engineering techniques such as directed evolution and structure-based design aim to improve the properties of natural proteins. The next step, the de novo insertion of function into previously inert protein scaffolds, is the lofty promise of computational protein design. In order to achieve this goal reliably and efficiently, computational methods can be iteratively improved by cycling between theory and experiment.
\r\nEfforts to both accelerate the rate and broaden the information exchanged within protein design cycles form the core of this thesis. Improvements in the throughput of experimental stability determination allowed the thorough assessment of new multi-state and library design tools. Intending to alleviate the fixed backbone, single native state design approximation, the study found constrained molecular dynamics ensembles useful for core repacking applications. The subsequent development of automated liquid handling protocols for common molecular biology techniques brings design experiments to new levels of sample throughput. This technology facilitated the creation of a stability database encompassing every single mutant in a small protein domain. Although constructed to facilitate future computational training efforts, we answer a multitude of questions pertaining to mutational outcomes, distributions, positional sensitivity, tolerance, and additivity in the context of a protein domain.
\r\nBy expanding the constraints of experimental molecular biology, this work opens up new possibilities in the efforts to train and assay new computational methodologies for protein engineering applications.
\r\n", "doi": "10.7907/Z9833Q2F", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6852", "collection": "thesis", "collection_id": "6852", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03172012-160452929", "primary_object_url": { "basename": "Romero_dissertation.pdf", "content": "final", "filesize": 6124840, "license": "other", "mime_type": "application/pdf", "url": "/6852/1/Romero_dissertation.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Statistical Models of the Protein Fitness Landscape: Applications to Protein Evolution and Engineering", "author": [ { "family_name": "Romero", "given_name": "Philip Anthony", "orcid": "0000-0002-2586-7263", "clpid": "Romero-Philip-Anthony" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Understanding the protein fitness landscape is important for describing how natural proteins evolve and for engineering new proteins with useful properties. This mapping from protein sequence to protein function involves an extraordinarily complex balance of numerous physical interactions, many of which are still not well understood. Directed evolution circumvents our ignorance of how a protein\u2019s sequence encodes its function by using iterative rounds of random mutation and artificial selection. The selection criteria is based on experimental measurements, which permits the optimization of protein sequence properties that are not understood. While directed evolution has been useful for exploring protein fitness landscapes, these searches have been relatively local in comparison to the vast space of possible protein sequences. Here, we present several classes of statistical models that map protein sequence space on a larger scale. We use these simple models to interpret data from SCHEMA recombination libraries, understand the evolutionary benefit of intragenic recombination, and design optimized protein sequences. By training on directly on experimental data, these models implicitly capture the numerous and possibly unknown factors that shape the protein fitness landscape. This provides an unrivaled quantitative accuracy across a massive number of protein sequences.
\r\n", "doi": "10.7907/7W9R-Y338", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7024", "collection": "thesis", "collection_id": "7024", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05112012-140027276", "primary_object_url": { "basename": "ThesisSJohnson2012_FINAL.pdf", "content": "final", "filesize": 42153918, "license": "other", "mime_type": "application/pdf", "url": "/7024/1/ThesisSJohnson2012_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "DNA Mechanics and Transcriptional Regulation in the E. coli lac Operon", "author": [ { "family_name": "Johnson", "given_name": "Stephanie Lynn", "clpid": "Johnson-Stephanie-Lynn" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Many gene regulatory motifs in both prokaryotes and eukaryotes involve physical manipulations of the genetic material, often on length scales short enough that the mechanical properties of the DNA significantly impact gene expression. One class of such manipulations, called \u201caction at a distance\u201d, includes transcription factor-mediated DNA looping, in which a binding site some distance away on the DNA is brought into close proximity with the transcription machinery at the promoter. DNA looping is a key component of several important regulatory systems in bacteria, and is crucial to the combinatorial control that is common at eukaryotic promoters regulated by more transcription factors than can physically bind adjacent to the promoter. Here we use a prototypical DNA looping protein, the Lac repressor from E. coli, to explore questions regarding the role of DNA mechanics in DNA looping and combinatorial control, particularly concerning the role of sequence flexibility in short-length-scale looping. We combine a statistical mechanical model of looping by the Lac repressor with a single-molecule technique called tethered particle motion that allows us to quantify this looping, and the systematic tuning of four biologically relevant and experimentally tractable parameters: loop length, loop sequence, repressor-DNA affinity, and repressor concentration. We show that this combination is a powerful approach to measuring repressor-DNA binding affinities and sequence-dependent DNA flexibilities in a way that is orthogonal, and therefore complementary, to conventional ensemble assays. Our results show that the sequence dependence to looping is more complicated than has been observed in other contexts, suggesting that \u201csequence flexibility\u201d as a general term is misleading, and, we argue, that the measurement of sequence flexibilities depend more strongly than previously appreciated on the shape of the deformation used to make the measurement. Finally, we present preliminary results with a more complicated system that is a case study for broader issues in combinatorial control, and a new hidden Markov model approach, based on variational Bayesian inference, to analyze these more complicated systems, which we hope will allow more precise dissections of, and more robust extraction of kinetic parameters from, tethered particle motion assays.", "doi": "10.7907/W40T-PD39", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7082", "collection": "thesis", "collection_id": "7082", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282012-103610958", "type": "thesis", "title": "Protein Folding and Macromolecular Dynamics: Fundamental Limits of Length and Time Scales", "author": [ { "family_name": "Lin", "given_name": "Milo Miaoyu", "clpid": "Lin-Milo-Miaoyu" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "thesis_committee": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Cross", "given_name": "Michael Clifford", "clpid": "Cross-M-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tombrello", "given_name": "Thomas A.", "clpid": "Tombrello-T-A" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "In this thesis, physics-based models of protein folding at the secondary and tertiary level are developed to resolve long-standing issues of protein folding kinetics. As discussed in the Introduction, the main objective is to provide fundamental limits on the length and time scales involved in protein folding. Protein folding is also placed within the broader context of macromolecular dynamics, which is extensively studied in the unfolded, folded, and unfolding regimes for the key molecular motifs of cellular biochemistry, including lipids, nucleic acids, and proteins. The effect of the water hydration and temperature are systematically probed to elucidate the crucial role of the environment in macromolecular stability and dynamics. For a wide range of bio-molecular phenomena, the observed collective behavior is shown to arise directly from first principles. Throughout, the emphasis is on analytic results free of tunable parameters, supported by ensemble-converging computational simulations, and corroborated by experimental evidence. ", "doi": "10.7907/CSW5-WY69", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6247", "collection": "thesis", "collection_id": "6247", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02162011-180032183", "primary_object_url": { "basename": "Chang_Ho_Sohn_PhD_Thesis_16May11_final.pdf", "content": "final", "filesize": 7745538, "license": "other", "mime_type": "application/pdf", "url": "/6247/7/Chang_Ho_Sohn_PhD_Thesis_16May11_final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "New Reagents and Methods for Mass Spectrometry-based Proteomics Investigations", "author": [ { "family_name": "Sohn", "given_name": "Chang Ho", "clpid": "Sohn-Chang-Ho" } ], "thesis_advisor": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" } ], "thesis_committee": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "New chemical reagents and methods have been employed for mass spectrometry (MS)-based proteomics investigations. Many chemical reagents are synthesized to be covalently attached to biomolecules, especially peptides and proteins. The properties of the resulting peptide conjugates are characterized by various tandem mass spectrometric techniques (e.g., collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), and free radical initiated peptide sequencing (FRIPS)). In Chapter 2, the effect of high electron affinity tags in ECD and ETD is investigated using their peptide conjugates. The initial intramolecular electron transfer from the high-lying Rydberg states to the covalently attached high electron affinity tag occurs in competition with the Coulomb stabilized \u03c0* orbitals of the amide bonds in the model peptides. This leads to the inhibition of the normal sequence of ECD and ETD processes, yielding no backbone fragmentations. In Chapter 3, selective disulfide bond cleavages are observed by the FRIPS method. A newly prepared TEMPO-based FRIPS reagent is labeled to model peptides containing disulfide bonds and subject to CID to monitor free radical induced cleavages. Highly selective C\u2013S and S\u2013S bond cleavages are observed and their reaction mechanisms are proposed. In Chapter 4, novel Caltech isobaric tags (CITs) for protein quantification are developed and validated using various model samples. A newly discovered low-energy gas-phase fragmentation pathway, a nucleophilic substitution of the N3 in the 1,2,3-triazole ring generated by copper-catalyzed azide-alkyne cycloaddition (CuAAC) inspired us to create CITs. This selective cleavage is applied to the formation of the reporter ions to quantify protein expression level in cells. Chapter 5 describes clickable cross-linkers (CXLs) developed for elucidation of three-dimensional protein structures and protein-protein interactions (PPIs). In CXLs, cross-linking reactions are separated from the conjugation of affinity tags, avoiding steric hindrance. Cross-linked peptides are enriched from the complex mixture of yeast lysate and cross-linked ubiquitin digests using avidin affinity chromatography, showing high sensitivity of the CXL-based analysis. The low-energy pathway used for CIT reagents is also adopted to produce the reporter ion, filtering MS/MS scans of cross-linked peptides from those of unmodified peptides.", "doi": "10.7907/HRF2-FJ21", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6509", "collection": "thesis", "collection_id": "6509", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082011-080856211", "primary_object_url": { "basename": "JAPS_Thesis.pdf", "content": "final", "filesize": 9905191, "license": "other", "mime_type": "application/pdf", "url": "/6509/1/JAPS_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Single-Molecule Studies of Ion Channels Expressing Unnatural Amino Acids", "author": [ { "family_name": "Shanata", "given_name": "Jai Anand Pattur", "clpid": "Shanata-Jai-Anand-Pattur" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The nicotinic acetylcholine receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission in the brain and peripheral nervous system. After an introduction (Chapter 1), Chapter 2 describes my development of techniques to combine single-channel and whole-cell recording with nonsense suppression. Having established the feasibility of the combined use of single-channel and whole-cell recording, in Chapter 3 we developed a method to identify the functional interactions of amino acids that are physically far apart in a protein. This is fundamentally a whole-cell recording method to find allosteric interactions in ion channels. The significance of this method is strongly supported by single-channel measurements. Additionally, the relationship between the single-channel gating equilibrium constant, theta, and the whole-cell measurement of EC50 is considered.
\r\n\r\nIn Chapter 4, I describe my progress towards measuring the channel opening rate of the fetal and adult muscle-type nicotinic acetylcholine receptors. Multiple different agonists are used, including acetylcholine, choline, and tetramethylammonium. Single-channel data are reported for the wild-type receptors as well as for receptors with the unnatural amino acid 5-F-Trp (monofluoro-Trp). Data are reported for multiple concentrations for a mutated fetal nAChR, and QuB is used to fit various possible models and estimate theta for this mutant.
\r\n\r\nA major aim of this dissertation was to use single-molecule studies of ion channels expressing unnatural amino acids to provide even more convincing evidence for cation-pi interactions at the binding sites of ligand-gated ion channels, specifically the neuronal nicotinic acetylcholine receptor. Chapter 5 describes the combined application of single-channel, whole-cell, and unnatural amino acid mutagenesis to the specific question of how two molecules\u2014nicotine and Chantix\u00ae (varenicline)\u2014bind to the alpha4beta2 brain receptor. In Chapter 6, I describe single-channel experiments that establish a method for distinguishing between the two known stoichiometries of the wild type alpha4beta2 brain receptor. Specifically, I identify a difference in the rectification properties of the high and low affinity receptors.
\r\n", "doi": "10.7907/2Y28-RP86", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6277", "collection": "thesis", "collection_id": "6277", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04032011-125158842", "primary_object_url": { "basename": "Complete_Thesis.pdf", "content": "final", "filesize": 6687006, "license": "other", "mime_type": "", "url": "/6277/12/Complete_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transfer Between [4Fe-4S] Cluster Glycosylases", "author": [ { "family_name": "Romano", "given_name": "Christine Anne", "clpid": "Romano-Christine-Anne" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work performed herein describes three proteins: Uracil DNA glycosylase (UDG) from Archaeoglobus fulgidus, MutY, and Endonuclease III (EndoIII) from Escherichia coli. They are DNA repair glycosylases that contain [4Fe-4S] clusters. While the catalytic mechanisms of many BER enzymes have been studied in detail, questions remain about how these enzymes search the vast amount of cellular DNA to find their substrates, and why some require a [4Fe-4S] cluster. The iron-sulfur cluster is not necessary for catalysis, and it only displays a physiologically relevant midpoint potential when bound to DNA. We have proposed that UDG, MutY, and EndoIII use their [4Fe-4S] clusters to participate in DNA-mediated charge transport (CT), and that these proteins mediate long-range electrochemical signaling in order to detect DNA damage.
\r\n\r\nThis scheme for DNA damage detection assumes that CT occurs efficiently between the DNA helix and the [4Fe-4S] cluster of the bound protein. In order for efficient CT to occur, a pathway of amino acids must be present that facilitates CT between the DNA and the iron-sulfur cluster. For each of the enzymes mentioned, this pathway was explored through mutagenesis. In UDG, MutY, and EndoIII, several amino acids thought to be important for CT were mutated and the resulting proteins were characterized biochemically. Their CT capabilities were analyzed by cyclic voltammetry on DNA-modified electrodes. In these experiments, the mutants\u2019 signal intensities were quantified and compared to those of wild-type enzyme. An attenuated signal relative to wild-type protein may indicate that the mutant is deficient in CT and that the targeted amino acid is part of the protein-DNA CT pathway in the native enzyme. Many mutants were also screened by enzymatic assays and circular dichroism spectroscopy to further characterize their DNA-binding properties and structural stability.
\r\n\r\nThe A. fulgidus UDG mutants examined, C17H, C85S, and C101S, all contained mutations in the cysteine residues that ligate the [4Fe-4S] cluster. These mutants were designed to determine how the iron-sulfur cluster coordination environment affects protein-DNA CT. The mutants exhibited varying signal strengths relative to WT UDG on DNA-modified electrodes. C85S produced a weaker signal, indicating a CT deficiency. The signal intensity from C101S was within error of that of WT, and the signal from C17H was larger than that of WT, possibly indicating that this mutant is less structurally stable than WT UDG.
\r\n \r\nIn E. coli MutY, position Y82 aligns with Y165 in MUTYH, a residue in which mutations have been found in many colorectal cancer patients. To better understand the correlation between protein-DNA CT and colorectal cancer, the MutY mutants Y82C and Y82L were prepared and characterized. Y82C exhibited a CT deficiency relative to WT MutY, whereas Y82L did not. These data indicate that Y82 forms part of the CT pathway in native E. coli MutY, but that other long-chain amino acids, such as leucine, can also mediate CT efficiently at this position.
\r\n \r\nSeveral different mutants of E. coli EndoIII were examined. First, the Y82 position was targeted, since the aligning MUTYH residue has been found mutated in colorectal cancer patients and because this residue is located near the protein-DNA interface. Five mutations were made at or near the Y82 position, and their cyclic voltammetry signals demonstrated that aromatic amino acids best mediate CT at this position. Other residues towards the interior of the protein, Y75, Y55, and F30 were also mutated to alanines. These mutants exhibited CT deficiencies, implicating the residues as part of a potential CT pathway. Residues W178 and Y185, located near the [4Fe-4S] cluster of EndoIII, were also mutated to alanines. The resulting mutants produced larger signals than that of WT EndoIII. These mutants were later shown by circular dichroism spectroscopy to be less stable structurally than WT EndoIII. All of the mutants mentioned exhibited enzymatic properties similar to those of WT, suggesting that they are able to bind DNA and excise damage nucleobases as well as the native enzyme. Several of these mutants were also used in a mutagenesis-based experiment to assay how EndoIII variants help MutY search for DNA lesions, although data from these experiments showed no significant differences in mutation rate between strains expressing different EndoIII variants.
\r\n \r\nIn total, the mutagenesis studies performed here helped determine the characteristics of BER enzymes that enable them to mediate DNA-protein CT. All these enzymes must contain a stable, well-protected metallocluster that charge can access through a series of CT-facilitating amino acids. In discovering several residues important for protein-DNA CT in UDG, MutY, and EndoIII, we have strengthened support for the hypothesis that these enzymes facilitate DNA-mediated CT in vivo. These enzymes may in fact be part of a much larger array of redox-active DNA-binding proteins that communicate electrochemically to help each other detect and repair DNA lesions inside the cell.
\r\n", "doi": "10.7907/63TC-FN74", "publication_date": "2011-06-10", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6375", "collection": "thesis", "collection_id": "6375", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05042011-174816761", "primary_object_url": { "basename": "CAWthesis.pdf", "content": "final", "filesize": 24168443, "license": "other", "mime_type": "application/pdf", "url": "/6375/9/CAWthesis.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Tuning Nitric Oxide Synthase: Investigating the Thiolate \"Push\" and No Release", "author": [ { "family_name": "Whited", "given_name": "Charlotte A.", "clpid": "Whited-Charlotte-A" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "All heme thiolate enzymes have conserved hydrogen bonding networks surrounding the axial thiolate ligand. In order to understand the role of this proximal hydrogen bonding network in nitric oxide synthases (NOS), three mutants of the NOS enzyme from Geobacillus stearothermophilus were expressed and characterized. The wild type enzyme has a tryptophan residue at position 70 that \u03c0-stacks with the porphyrin ring and donates a long hydrogen-bonding interaction to the thiolate ligand of the heme iron. The native Trp was replaced with His, Phe, and Tyr. These three residues were selected to investigate the two effects of the Trp, H-bonding and Pi-stacking. Several different spectroscopic techniques were used to investigate the stability and properties of these mutant enzymes. The identity of each mutant was confirmed by mass spectrometry. Both UV-visible absorption and circular dichroism spectroscopies were used to assess the stability of the new proteins. It was shown using binding assays, generation of the ferrous-CO species, and redox titrations that the \u03c3-donating abilities of the thiolate are increased after removal of the hydrogen bonding group in the Trp. Finally, electron paramagnetic resonance spectroscopy and Evans method nuclear magnetic resonance spectroscopy were used to characterize the spin state of the iron center in each mutant, reflecting the increased \u03c3-donating capabilities of the thiolate upon removal of the hydrogen bonding group. The reduction potential of wild type and W70H were determined by chemical titration to be -362 and -339 mV vs. NHE, respectively. This is the first report of the reduction potential of any bacterial nitric oxide synthase.
\r\n\r\nThe reactivity of each the wild type enzyme and the three new mutants was tested using stopped-flow mixing coupled with UV-visible absorption spectroscopy and the Griess Assay. Autoxidation rates measured by stopped-flow suggest that the Tyr and Phe mutants do indeed have significantly more negative reduction potentials, but that the His mutant is particularly slow to oxidize. The Griess Assays showed that all four enzymes produce nitrite in solution, when provided with substrate, cofactor and hydrogen peroxide (as a source of reducing equivalents). In single turnover experiments, however, only three of the four enzymes showed evidence of ferric-NO production. The His mutant showed no intermediate absorbance near 440 nm (which would be indicative of ferric-NO formation), suggesting that it releases NO- rather than the radical species NO\u2219. The role of this hydrogen bond is concluded to be an electronic one, rather than playing any part in positioning the heme. It prevents formation of the inactive P420 species, and tunes the reduction potential to one high enough to be reduced by a reductase but low enough to still deliver an electron to the redox active cofactor, tetrahydrobiopterin, at the end of catalysis.
\r\n\r\nThe rate at which NO is released by each NOS enzyme varies greatly among isoforms and species, over nearly two orders of magnitude. One residue (an isoleucine located above the heme in bacterial enzymes) involved in the gating of NO release has been previously identified by Stuehr. However, this single residue does not account for the entirety of the differences among the forms of NOS. Another residue, a histidine at position 134 in NOS from Geobacillus stearothermophilus (gsNOS), was hypothesized to also participate in gating NO release based on an observed correlation between rates of NO release and the bulk of side chains at this position. Each single point mutation, H134S and I223V, and the double mutant were expressed in gsNOS and their reactivity toward the diatomic molecules CO and NO were studied. CO rebinding was investigated using laser flash photolysis and NO release using stopped flow UV-visible spectroscopy. The presence of both monomer and dimer was observed in solution, and position 134 was shown to be another key residue in gating NO release. Wild type gsNOS contains both the bulkier Ile223 and His134 and has the slowest measured NO release (0.039 s-1) of all NOS enzymes. A new, more accurate kinetics model for turnover is proposed. Each single mutation increased NO release substantially, while the double mutant has a rate constant of 1.0 s-1, nearly as fast as mammalian iNOS at 2.3 s-1, identifying position 134 as another important factor determining rate constants for NO release.
\r\n", "doi": "10.7907/PZAY-WQ64", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6231", "collection": "thesis", "collection_id": "6231", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01242011-170306838", "primary_object_url": { "basename": "Saouma_final.pdf", "content": "final", "filesize": 12129295, "license": "other", "mime_type": "application/pdf", "url": "/6231/1/Saouma_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Iron Mediated Reduction Schemes for Dinitrogen and Carbon Dioxide", "author": [ { "family_name": "Saouma", "given_name": "Caroline Thalia Abdunnur", "clpid": "Saouma-Caroline-Thalia-Abdunnur" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Several mono- and diiron species that coordinate NxHy ligands have been prepared and studied, to serve as structural, spectroscopic, and/or reactivity mimics to intermediates to an alternating reduction scheme for N\u2082 (i.e., Mn-N\u2261N \u2192 Mn-HN=NH \u2192 Mn-H\u2082N-NH\u2082 \u2192 Mn + 2 NH\u2083). The reaction between [PhBPR\u2083]FeMe ([PhBPR\u2083] = (PhB(CH\u2082PR\u2082)\u2083-; R = Ph, CH\u2082Cy) and hydrazine affords {[PhBPR\u2083]Fe}\u2082(\u03bc-\u03b7\u00b9: \u03b7\u00b9-N\u2082H\u2084)(\u03bc\u00b2- \u03b7\u00b2:N\u2082H\u2082). In one instance (R = Ph), the stepwise oxidation of coordinated hydrazine to diazene, and diazene to dinitrogen is achieved, giving {[PhBPPh\u2083]Fe}\u2082(\u03bc-\u03b7\u00b9:\u03b7\u00b9-N\u2082H\u2082)(\u03bc-\u03b7 2: \u03b7 2-N2H2) and {[PhBPPh\u2083]Fe}\u2082(\u03bc-NH)\u2082, respectively.
\r\n\t\r\nAs an extension to this work, a family of complexes which feature the same auxiliary ligands (i.e., [PhBPCH2Cy\u2083]Fe(OAc)), that are all iron(II), and that only differ in the oxidation state of the nitrogenous ligand has also been prepared: {[PhBPCH2Cy\u2083]Fe(OAc)}\u2082(\u03bc-N\u2082), {[PhBPCH2Cy\u2083]Fe(OAc)}\u2082(\u03bc-N\u2082H\u2082), {[PhBPCH2Cy\u2083]Fe(OAc)}\u2082(\u03bc-N\u2082H\u2084), and {[PhBPCH2Cy\u2083]Fe(OAc)(NH\u2083).
\r\n\t\r\nTo determine whether similar species could be isolated at a single iron site, the coordination chemistry of the more crowded \u201c[PhBPmter3]Fe\u201d fragment was investigated and compared to that of the \u201c[PhBPPh3]Fe\u201d scaffold. Treatment of [PhBPmter3]FeMe with hydrazine generates the unusual 5-coordinate hydrazido complex, [PhBPmter3]Fe(\u03bc2-N2H3), which features an Fe=N \u03c0 bond. Both 5- and 6-coordinate iron complexes that coordinate hydrazine were also synthesized, and the oxidation of these hydrazine and hydrazido(-) species was explored. In most instances, oxidation results in disproportionation of the N2Hy ligand, and [PhBPR3]Fe(NH3)(OAc) (R = Ph, mter) is isolated.
\r\n\t\r\nA 5-coordinate diiron diazene redox pair of complexes, {[PhBPPh3]Fe(CO)}2(\u03bc-\u03b71:\u03b71-N2H2)0/- was also prepared and studied. The electronic structure of the Fe-NH-NH-Fe core in these complexes is unusual in that it features a highly activated diazene ligand, which is unprecedented for mid-to-late transition metals. Combined structural, spectroscopic, and computation studies indicate that there is much \u03c0-covalency within the Fe-NH-NH-Fe core, which has a similar electronic structure as butadiene.
\r\n\t\r\nWith regards to CO2 reduction, the ability of iron(I) to mediate the one- and two- electron reductions of CO2 was explored. The reaction between [PhBPCH2Cy3]Fe(PCy)3 and CO2 is solvent dependent, with oxalate formation to generate {[PhBPCH2Cy3]Fe}2(\u03bc-\u03b72:\u03b72-oxalato) being favored in THF, and decarbonylation to give {[PhBPCH2Cy3]Fe}2(\u03bc-O)(\u03bc-CO) occurring exclusively in MeCy. Studies aimed at understanding this unusual solvent-induced selectivity are presented.
\r\n", "doi": "10.7907/46C3-BY97", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6429", "collection": "thesis", "collection_id": "6429", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242011-165030719", "primary_object_url": { "basename": "Thesis_Peera.pdf", "content": "final", "filesize": 39277548, "license": "other", "mime_type": "application/pdf", "url": "/6429/9/Thesis_Peera.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Post-Translational Membrane Protein Targeting by the Chloroplast Signal Recognition Particle", "author": [ { "family_name": "Jaru-Ampornpan", "given_name": "Peera", "orcid": "0000-0002-4610-2235", "clpid": "Jaru-Ampornpan-Peera" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Post-translational transport of membrane proteins poses enormous challenges to the cells. The transport factors must accurately select and deliver the cargos to the appropriate target membranes. In addition, they have to provide chaperone for their hydrophobic cargos. To understand capacity and limitation of a post-translational transport factor, we studied one of the most efficient membrane protein transport pathways, the delivery of light-harvesting chlorophyll-binding (LHC) proteins to the thylakoid membrane. This targeting reaction is mediated by the chloroplast Signal Recognition Particle (cpSRP) and its receptor. Although the core SRP GTPases are close homologues of those in cytosolic SRP pathways, the unique features of cpSRP that might reflect its adaptation to the challenges in post-translational targeting include (i) the lack of the otherwise universally conserved SRP RNA, and (ii) the exclusive presence of a novel protein, cpSRP43. In the first part of this thesis, we define the thermodynamic and kinetic framework for the GTPase cycles of cpSRP and its receptor and uncover the molecular bases that enable their intrinsically fast interactions, such that they can bypass an SRP RNA, an essential accelerator for the cytosolic SRP\u2013receptor interaction. The second part of the thesis is devoted to characterization of the chaperone function of cpSRP43. We show that cpSRP43 specifically and effectively prevents and reverses the aggregation of its cargo, LHC proteins. We further investigate the molecular mechanism of this novel disaggregase activity, using a combination of biochemical and structural approaches. In summary, this dissertation aims to understand how cpSRP and its receptor adapt to their unique requirements in efficiently transporting a family of highly abundant membrane proteins. ", "doi": "10.7907/RDKZ-8094", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6204", "collection": "thesis", "collection_id": "6204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12142010-165109760", "primary_object_url": { "basename": "Muzikar_Thesis.pdf", "content": "final", "filesize": 10132799, "license": "other", "mime_type": "application/pdf", "url": "/6204/8/Muzikar_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Repression of DNA-Binding-Dependent Glucocorticoid Receptor-Mediated Gene Expression", "author": [ { "family_name": "Muzikar", "given_name": "Katy Ann", "clpid": "Muzikar-Katy-Ann" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Gene expression is controlled by transcription factors that regulate the rates at which genes are expressed either by recruiting or inhibiting protein complexes that bind to the promoters or enhancers of target genes. Molecules that can specifically modulate these protein-DNA interfaces show promise as tools for understanding gene regulation pathways and may have application in human medicine. Hairpin pyrrole-imidazole polyamides are programmable oligomers that bind the DNA minor groove in a sequence-specific manner with affinities comparable to those of natural DNA-binding proteins. These cell-permeable small molecules have been shown to enter the nuclei of live cells, disrupt protein-DNA interactions, and downregulate endogenous gene expression. This thesis describes the use of polyamides to modulate gene expression in order to probe gene regulation mechanisms of several different biologically relevant systems. A polyamide is designed to target the glucocorticoid receptor transcription factor DNA binding site located in the promoter of the glucocorticoid-induced leucine zipper gene. This polyamide is shown to bind with high affinity to the promoter sequence, modulate the expression of this gene, and disrupt the binding of the protein to the gene\u2019s promoter. Examination of the global effects of this polyamide on mRNA transcription is used to elucidate a list of genes that are regulated by a glucocorticoid receptor protein-DNA dependent mechanism. Also in this thesis, the specificities of a Cy3-labeled polyamide known to downregulate expression of the Vascular Endothelial Growth Factor is examined using DNA microarrays composed of hairpins harboring all 524,800 unique 10 base pair DNA sequences. We experimentally verify the correlation of Cy3 fluorescence intensity with quantitative DNase I footprint-derived binding affinities. Additionally, progress is made towards the polyamide-mediated inhibition of Myc/Max transcription factor gene regulation. ", "doi": "10.7907/FKCV-KP50", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6156", "collection": "thesis", "collection_id": "6156", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10242010-143824484", "primary_object_url": { "basename": "Full_thesis.pdf", "content": "final", "filesize": 98888164, "license": "other", "mime_type": "application/pdf", "url": "/6156/9/Full_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Biochemical and Biophysical Characterizations of Immunoglobulin Superfamily Receptors Neogenin and L1", "author": [ { "family_name": "Yang", "given_name": "Fan", "clpid": "Yang-Fan" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Kuppermann", "given_name": "Aron", "clpid": "Kuppermann-A" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Immunoglobulin (Ig) superfamily receptors function in a wide variety of developmental and metabolic processes. We are particularly interested to characterize two Ig superfamily receptors neogenin and L1. The first chapter of the thesis gives a brief review of the biological significance of neogenin and L1 and what has been learned in their functions. In Chapter 2, we described the localization of the hemojuvelin-binding epitope of neogenin to the membrane proximal fifth and sixth fibronectin type III (FNIII) domains, with the sixth FNIII domain contributing the majority of the binding. Chapter 3 presents the crystal structure of this hemojuvelin-binding fragment at 1.8 \u00c5, revealing a nearly linear domain arrangement. Hemojuvelin binding sites have been mapped to one face of the sixth FNIII domain based on sequence alignment between neogenin and DCC (Deleted in Colorectal Cancer), a molecule related to neogenin but does not bind to hemojuvelin. These results should also be informative in understanding the interaction between neogenin and repulsive guidance molecule (RGM), the closest homologue of hemojuvelin. The interaction between neogenin and RGM is known to regulate neuronal survival. Chapter 4, the second part of the thesis, describes our studies of L1-mediated homophilic adhesion using biophysical approaches. We built a basis shape model to describe L1-mediated homophilic adhesion between L1-coated giant unilamellar vesicles and flat substrate. Using confocal microscopy techniques, we were able to reconstruct the three-dimensional shape of an adhered vesicle. We developed an algorithm in order to derive adhesion strength from the configurations of adhered vesicles based on our basis shape model using energy minimization approach.", "doi": "10.7907/Q8ET-K632", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6036", "collection": "thesis", "collection_id": "6036", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09192010-204534950", "primary_object_url": { "basename": "Bugg_CW_Thesis_Final.pdf", "content": "final", "filesize": 18920297, "license": "other", "mime_type": "application/pdf", "url": "/6036/1/Bugg_CW_Thesis_Final.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Domain Organization of Mutant Huntingtin Fibrils", "author": [ { "family_name": "Bugg", "given_name": "Charles Walter", "clpid": "Bugg-Charles-Walter" } ], "thesis_advisor": [ { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Langen", "given_name": "Ralf", "clpid": "Langen-R" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Langen", "given_name": "Ralf", "clpid": "Langen-R" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Huntington\u2019s disease is a progressive, fatal neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the huntingtin gene (HDx1). A hallmark of the disease is the formation of fibrillar aggregates within cells. In vitro, HDx1 with a polyQ expansion forms fibrils that have a cross beta structure common to amyloid fibrils, but little else is definitively known about HDx1 fibril structure. We used electron paramagnetic resonance spectroscopy to study the organization of the major domains (N-terminus, polyQ, C-terminus) of HDx1 with 46Q within the fibril. Our data show that HDx1 fibrils do not have a parallel, in-register structure like most other disease-associated amyloid fibrils. The C-terminus is highly dynamic and is attached like a tail to the polyQ domain, which is mostly immobilized and forms the core of the fibril. However, the C-terminal portion of the polyQ lies outside the core and has a mobility similar to the C-terminus. The N-terminus produced heterogeneous spectra, indicating that it is able to sample multiple conformations. In sum, our study excluded the parallel, in-register arrangement of beta strands within HDx1 fibrils and represents a first step toward a high-resolution structure of HDx1 fibrils.", "doi": "10.7907/XAYY-9E20", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5997", "collection": "thesis", "collection_id": "5997", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08182010-104705359", "primary_object_url": { "basename": "Full_Dissertation_(N._Ballor).pdf", "content": "final", "filesize": 13949251, "license": "other", "mime_type": "application/pdf", "url": "/5997/1/Full_Dissertation_(N._Ballor).pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Hydrogenases and Hydrogen Sensors in the Symbiotic Microbial Communities of Wood-Feeding Termites", "author": [ { "family_name": "Ballor", "given_name": "Nicholas R.", "clpid": "Ballor-Nicholas-R" } ], "thesis_advisor": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "thesis_committee": [ { "family_name": "Orphan", "given_name": "Victoria J.", "clpid": "Orphan-V-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Mazmanian", "given_name": "Sarkis", "clpid": "Mazmanian-S-K" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The termite gut is an ideal ecosystem for studying hydrogen ecophysiology. Hydrogen is central to the obligate mutualism between termites and their gut microbes and is turned over at rates as high as 33 m3 H2 per m3 hindgut volume daily and maintained near saturation in some species. Acetogenic bacteria use hydrogen to produce up to 1/3 of the total flux of the termite\u2019s primary carbon and energy source, acetate. We have taken a three-fold approach to investigate the hydrogen ecophysiology of the termite gut. In our first approach (Chapter 2) we completed a bioinformatic analysis of [FeFe] hydrogenase-like (H domain) proteins encoded in the genomes of three termite gut treponemes. Treponemes are among the most highly represented groups of gut bacteria. The remarkable diversity of H domain proteins encoded accentuates the importance of hydrogen to their physiology. Moreover, they encoded a poorly understood class hydrogen sensing H domain proteins and thereby present a unique opportunity for their further study. In our second approach (Chapters 3 and 4) we analyzed molecular inventories prepared from termite gut microbiomes of a class of [FeFe] hydrogenases found highly represented in a termite hindgut metagenome. The libraries of peptide sequences clustered with one another in a manner congruent with termite host phylogeny suggesting co-evolution. Interestingly, we observed that higher termite guts may harbor higher sequence diversity than lower termites. In our third approach (Chapter 5) we used microfluidic digital PCR to identify bacteria in the gut of Reticulitermes tibialis encoding [FeFe] hydrogenases. The majority of the 16S rRNA gene phylotypes observed to co-amplify with hydrogenase sequences were treponemal, and the only observed instances of the same 16S rRNA-hydrogenase gene pair co-amplifying in multiple microfluidic chambers corresponded to treponemal phylotypes. Therefore, treponemes may be an important or predominant bacterial group encoding an important family of [FeFe] hydrogenases in the termite gut. The above results provide support for an important role for treponemes in mediating hydrogen metabolism in the termite gut and accentuate the intimacy and stability of the association termites have maintained over the course of their evolution with their gut microbial communities. ", "doi": "10.7907/621E-9221", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5269", "collection": "thesis", "collection_id": "5269", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06162009-143222", "primary_object_url": { "basename": "FullThesis1.pdf", "content": "final", "filesize": 73920847, "license": "other", "mime_type": "application/pdf", "url": "/5269/11/FullThesis1.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Investigations into the Generality of Metalloinsertion at DNA Defects", "author": [ { "family_name": "Zeglis", "given_name": "Brian Matthew", "clpid": "Zeglis-Brian-Matthew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metalloinsertors are substitutionally inert, octahedral transition metal complexes that bind to thermodynamically destabilized mismatched sites in duplex DNA with high affinity and selectivity. The complexes approach DNA from the minor groove, eject the mismatched bases into the major groove, and replace the displaced bases in the helical \u03c0-stack with their own sterically expansive ligands. Herein, we describe a series of five investigations aimed at elucidating the generality of metalloinsertion at DNA defects.
\r\n\r\nIn an effort to develop a diagnostic for mismatched DNA, a bifunctional, mismatch-specific conjugate with rhodium metalloinsertor and fluorophore subunits has been constructed. A proof-of-concept conjugate was successfully produced that displays an almost fourfold fluorescence enhancement in the presence of mismatched versus matched DNA.
\r\n\r\nTo investigate the range of metal complexes capable of mismatch-specific metalloinsertion, a ruthenium bisdipyridyl complex bearing the heptacyclic eilatin ligand has been synthesized and characterized. Electrophoresis competition experiments illustrate that the complex does display mismatch-preferential, though not necessarily mismatch-selective, binding.
\r\n\r\nTo probe the generality of metalloinsertion at other common thermodynamically destabilized DNA defects, the binding of rhodium metalloinsertors at abasic sites and single base bulges has been studied. It was determined that metalloinsertors bind abasic sites with high affinity and specificity, without regard to the identity of the unpaired base and with little dependence on the sequence context of the defect. Single base bulge recognition proved more elusive, with both the identity of the unpaired base and the sequence context influencing recognition.
\r\n\r\nTo determine the structural generality of metalloinsertion, single crystal X-ray diffraction was employed to determine the structure of \u0394-Rh(bpy)2(chrysi)3+ bound to an oligonucleotide duplex containing two A\u2022A mismatches. Two structures were obtained at <2 \u00c5 resolution, and each provides an archetypical picture of metalloinsertion: the bulky rhodium complex inserts into the mismatched site from the minor groove, ejecting the mismatched bases and replacing the displaced base pair with its own sterically expansive ligand.
\r\n\r\nFinally, two mismatch-specific conjugates have been designed for chemotherapeutic applications: a metalloinsertor-oxaliplatin conjugate for the selective delivery of platinum chemotherapeutics to mismatch repair deficient cells and a metalloinsertor-Auger electron emitter conjugate for the selective irradiation of mismatch-containing DNA.
", "doi": "10.7907/0YRQ-3W54", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5855", "collection": "thesis", "collection_id": "5855", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272010-004616557", "primary_object_url": { "basename": "zhang_thesis_2010.pdf", "content": "final", "filesize": 27703957, "license": "other", "mime_type": "application/pdf", "url": "/5855/18/zhang_thesis_2010.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Multistate GTPases Control Cotranslational Protein Targeting", "author": [ { "family_name": "Zhang", "given_name": "Xin", "clpid": "Zhang-Xin" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The cotranslational protein targeting process transports roughly one-third of proteins in a cell\u2019s genome from the cytoplasmic space to the membrane compartments. This process is regulated by the signal recognition particle (SRP) and its receptor (SR). I aim to understand how the complex assembly and activation of GTP hydrolysis during the SRP-SR interaction are controlled so that the SRP machinery functions as a molecular switch to regulate the series of molecular events in space and time. Using a combination of biochemical and biophysical approaches, this dissertation has defined the kinetic and thermodynamic framework of the SRP-SR interaction and has elucidated the regulatory role of the SRP-SR interaction on the protein targeting process. In particular, this dissertation demonstrates that the function of the SRP machinery is governed by a series of ordered conformational changes during SRP-SR interaction that culminate in their activation of GTP hydrolysis. Further, these conformational changes closely monitor and actively respond to the biological cues so that they provide discrete control points at which regulation can be exerted on the protein targeting reaction spatially and temporally. The paradigm provided in this dissertation offers a mechanistic view of another fascinating system in which multistate protein machineries control critical biological processes with exquisite order.", "doi": "10.7907/28HP-FJ94", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5824", "collection": "thesis", "collection_id": "5824", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212010-154212167", "primary_object_url": { "basename": "thesis_VAN.pdf", "content": "final", "filesize": 1671048, "license": "other", "mime_type": "application/pdf", "url": "/5824/1/thesis_VAN.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Efficient Generation of Hyperpolarized Molecules Utilizing the Scalar Order of Parahydrogen", "author": [ { "family_name": "Norton", "given_name": "Valerie Ann", "clpid": "Norton-Valerie-Ann" } ], "thesis_advisor": [ { "family_name": "Weitekamp", "given_name": "Daniel P.", "clpid": "Weitekamp-D-P" } ], "thesis_committee": [ { "family_name": "Kuppermann", "given_name": "Aron", "clpid": "Kuppermann-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Weitekamp", "given_name": "Daniel P.", "clpid": "Weitekamp-D-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation describes methods that polarize the spin of a specific nucleus in molecules synthesized by molecular addition of parahydrogen to a precursor molecule. Nuclear magnetic resonance (NMR) pulse sequences are designed to perform efficient transfer of spin order by way of the scalar spin couplings between the two nascent protons and a heteronuclear spin label target. The result is an increase in the NMR signal from that nucleus by several orders of magnitude, approaching unity polarization. Algorithms are presented to effect the desired unitary evolution of this three-spin system over the range of couplings found in diverse molecules and in the presence of interfering spins. These methods are explored theoretically and comparisons are made to select the most advantageous method given a specific problem.
\r\n\r\nIssues concerning the choice of target molecule, portable equipment, and automation are discussed. Some design choices made for convenience in one aspect of the execution of the methods raise difficulties in other aspects. These difficulties are elucidated and methods of mitigation are discussed.
\r\n\r\nPulse design issues are elucidated with numerical calculations which confirm analytical results for the time dependence obtained in the multiply rotating frame approximation. Failures of this approximation at low frequencies are explored numerically leading to novel pulse sequence design rules which ameliorate undesirable phenomena peculiar to low field NMR, enabling its employment for this and other applications requiring precise control of the spin degrees of freedom. Experimental results, primarily aimed at biomedical applications, are reviewed.
\r\n", "doi": "10.7907/57FW-1060", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5790", "collection": "thesis", "collection_id": "5790", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05102010-102555148", "type": "thesis", "title": "Geochemical Mechanisms of Biomineralization from Analysis of Deep-Sea and Laboratory Cultured Corals", "author": [ { "family_name": "Gagnon", "given_name": "Alexander C.", "clpid": "Gagnon-Alexander-C" } ], "thesis_advisor": [ { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Eiler", "given_name": "John M.", "clpid": "Eiler-J-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ocean is a major component of global heat transport and represents a large exchangeable reservoir of CO\u2082. The importance of these effects on climate can be quantified with records of ocean temperature, chemistry and dynamics spanning past climate change. One approach to reconstruct past ocean conditions relies on the chemical composition of CaCO\u2083 skeletons from coral. Despite the utility of these geochemical proxies, several lines of evidence suggest that biomineralization, the process corals use to build their skeletons, also influences composition, complicating the interpretation of past records. Coral grown under constant environmental conditions, either collected from the deep-sea or cultured in the laboratory, are used to quantify and spatially map the effects of biomineralization on skeletal composition.
\r\n\r\nIn modern deep-sea coral, Mg/Ca increases with decreasing Sr/Ca in most the skeleton, consistent with closed-system (Rayleigh) precipitation. Results also show composition strongly follows skeletal architecture. Centers of calcification (COCs) are small regions of disorganized crystals thought to be the initial stage of skeletal extension. Unlike the rest of the skeleton, Mg/Ca ratios vary more than two fold within the COCs while Sr/Ca is near constant. Our data provide new constraints on a number of possible mechanisms for this effect.
\r\n\r\nIn a complementary set of experiments the nanoSIMS, a new instrument capable of accurate sub-micron compositional analysis, is applied to adult cultured surface coral (1) mapping the pattern of metal ion incorporation in new growth and showing that the calcifying fluid is likely in direct exchange with seawater; and (2) testing the sensitivity of Me/Ca ratios to aragonite saturation \u03a9. Despite a large range of \u03a9 and calcification rates, the average Sr/Ca of nanoSIMS spot measurements in cultured coral are within 1.2% (2 sigma std. dev. of the 5 means). These data suggest that temperature is a more significant control on Sr/Ca than aragonite saturation between \u03a9 = 2.5--5. Within the framework of a closed-system (Rayleigh) model for biomineralization the results constrain explanations for the sensitivity of coral calcification rates to ocean acidification, improving our understanding of how anthropogenic CO\u2082 will impact coral reefs.
", "doi": "10.7907/N1MW-8Q84", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5603", "collection": "thesis", "collection_id": "5603", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03142010-163350106", "primary_object_url": { "basename": "Kiowa_Bower_Thesis_2010.pdf", "content": "final", "filesize": 56594011, "license": "other", "mime_type": "application/pdf", "url": "/5603/1/Kiowa_Bower_Thesis_2010.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Chemical-Scale Studies of the 5-HT\u2083 and D2 Dopamine Receptors ", "author": [ { "family_name": "Bower", "given_name": "Kiowa San", "clpid": "Bower-Kiowa-San" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "During synaptic transmission in the central nervous system, neuroreceptors transduce a chemical signal into an electrical signal, a process that is mediated by both ligand-gated ion channels (LGICs) and G-protein coupled receptors (GPCRs). The work in this thesis examines structure-function relationships within these receptors, with a focus on elucidating the mechanism of molecular recognition during ligand binding. We utilize conventional and unnatural amino acid mutagenesis, structural derivatives of agonists, and homology models to identify specific interactions and the role of binding site residues in ligand binding and receptor activation. The technique of unnatural amino acid mutagenesis allows us to study these processes in greater detail than would otherwise be possible, even at the scale of a chemical bond.
\r\n\r\nChapter 2 covers structure-function investigations of a ligand-gated ion channel, the 5-HT\u2083 receptor, with a goal of understanding agonist binding and receptor activation. The project examines residues in close proximity to the ligand-binding site and focuses on polar interactions with hydrophilic residues. We identify 5-fluorotryptamine (5-FT) as a partial agonist of the 5-HT\u2083 receptors and show that size and electronegativity are important at the 5\u2019 position for efficient channel opening. Our investigation of the compound 1-OT revealed it to be an agonist of equal potency to the native agonist (5-HT), demonstrating that the indolic proton of serotonin is not essential to its activation of the receptor. A study focusing on loop A residues led us to refine our homology model and propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Further studies of binding site residues identified an ionic interaction that likely participates in the conformational changes associated with receptor gating and characterized several other residues that play critical roles in receptor activation. Finally, we compare and contrast the behaviors of two structurally distinct agonist classes, 5-HT and its related structures, and m-chlorophenylbiguanide (mCPBG) and identify several residues that play critical roles in modulating agonist binding and gating in response to these agonists.
\r\n\r\nChapter 3 describes a study examining the binding site and the mechanism of agonist activation of a GPCR, the D2 dopamine receptor. A number of aromatic amino acids thought to be near the agonist binding site were evaluated. Incorporation of a series of fluorinated tryptophan derivatives at a conserved tryptophan of the D2 receptor establishes a cation-\u03c0 interaction between the agonist dopamine and this residue (W6.48), suggesting a reorientation of W6.48 on agonist binding, consistent with proposed \"rotamer switch\" models.
\r\n\r\nFinally, chapter 4 describes a project that seeks to extend the nonsense suppression methodology to include mammalian expression systems. Progress is made developing techniques for efficient transfection of cells in culture.
\r\n", "doi": "10.7907/17AK-6J11", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5499", "collection": "thesis", "collection_id": "5499", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062010-122905478", "primary_object_url": { "basename": "CAP_thesis.pdf", "content": "final", "filesize": 11296575, "license": "other", "mime_type": "application/pdf", "url": "/5499/8/CAP_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "The Cellular Uptake of Luminescent Ruthenium Complexes", "author": [ { "family_name": "Puckett", "given_name": "Cindy Ann", "clpid": "Puckett-Cindy-Ann" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transition metal complexes have enormous potential as diagnostic and therapeutic agents, but their internalization and distribution in living cells are only poorly understood. Here, we perform one of the few systematic explorations of the uptake efficiency and mechanism of a class of metal complexes: luminescent dipyridophenazine (dppz) complexes of ruthenium(II). Substitution of the ancillary ligands permits variation in the overall complex charge, size, and hydrophobicity. We find that internalization of these complexes occurs mostly through passive diffusion, driven by the membrane potential, and that hydrophobicity, rather than size, is the most important determinant of compound accumulation. Across different cell types with all compounds, mostly uneven cytoplasmic staining is observed with near exclusion from the nucleus. Conjugation to cell-penetrating peptides, such as D-octaarginine, increases uptake efficiency, but leads to trapping in endosomes below a threshold concentration. Above this threshold concentration, substantial staining of the nucleus as well as the cytosol is observed. An appended fluorescein tag lowers the threshold concentration, indicating the importance of payload to the internalization and distribution of cell-penetrating peptides. Shorter peptides, including the nuclear targeting signal RrRK (where r = D-arginine), are also studied, though none have as high a degree of uptake nor as low a threshold concentration as the octaarginine conjugate. These studies provide a basis for the future design and optimization of metal complexes for biological application. ", "doi": "10.7907/2484-1405", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5489", "collection": "thesis", "collection_id": "5489", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01032010-225423037", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 12271311, "license": "other", "mime_type": "application/pdf", "url": "/5489/8/thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Contributions of Dna2 and the Tim/Tipin Complex to Genomic Stability", "author": [ { "family_name": "Wawrousek", "given_name": "Karen Elizabeth", "clpid": "Wawrousek-Karen-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Dunphy", "given_name": "William G.", "clpid": "Dunphy-W-G" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Dunphy", "given_name": "William G.", "clpid": "Dunphy-W-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "This thesis describes the essential roles of Dna2 and the Tim/Tipin complex in the maintenance of genomic stability. Dna2 participates in DNA replication and double-strand break repair by homologous recombination. Meanwhile, the Tim/Tipin complex is required for efficient checkpoint activation upon replication stress, which can be caused by stalled DNA replication forks.
\r\n\r\nWhile yeast genetics and experiments with purified proteins have revealed much about yeast Dna2, we chose to pursue characterization of metazoan Dna2 using Xenopus cell-free extracts. We show that binding of Dna2 to origins of replication is dependent upon formation of pre-replication complexes but independent of CDK2 activity. Upon initiation of DNA replication, Dna2 travels with replication forks. Physical interactions with Mcm10 and And-1, proteins involved in lagging strand DNA replication, are indicative of a role in replication of the lagging strand; this result is consistent with genetic results in yeast and in vitro biochemical experiments.
\r\n\r\nDna2 also participates in the response to double-strand breaks and accumulates on chromatin containing double-strand breaks. We show that Dna2 binds to free DNA ends after the Mre11-Rad50-Nbs1 complex and ATM, but before RPA. Dna2-depleted extracts exhibit delayed processing of DNA ends, indicating that other nucleases do not easily compensate for the lack of Dna2. Consistent with genetic results in yeast, we find that the Mre11-Rad50-Nbs1 protein complex is essential for the processing of free DNA ends, but inhibition of Mre11 nuclease activity only slows processing. This observation indicates that other nucleases, possibly Dna2, can compensate for loss of Mre11 nuclease activity. Despite the role of Dna2 in double-strand break processing, Dna2 is not required for checkpoint activation.
\r\n\r\nTimeless (Tim) and Tipin participate in the checkpoint response to stalled replication forks. We demonstrate here that Tim and Tipin form a complex, associate with chromatin in S phase, and physically interact with many proteins at the replication fork. Human cells lacking the Tim/Tipin complex do not exhibit robust checkpoint activation in response to stalled replication forks. Finally, we show that Tipin is also a target of both the ATR and Cdc7 kinases, which respond to stalled replication forks.
\r\n", "doi": "10.7907/QYSH-WH15", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5245", "collection": "thesis", "collection_id": "5245", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08072008-171049", "primary_object_url": { "basename": "PeterLeongThesis.pdf", "content": "final", "filesize": 7198483, "license": "other", "mime_type": "application/pdf", "url": "/5245/7/PeterLeongThesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Computational Challenges in High-Resolution Cryo-Electron Microscopy", "author": [ { "family_name": "Leong", "given_name": "Peter Anthony", "clpid": "Leong-Peter-Anthony" } ], "thesis_advisor": [ { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Zhou", "given_name": "Z. Hong", "clpid": "Zhou-Z-Hong" }, { "family_name": "Fultz", "given_name": "Brent T.", "orcid": "0000-0002-6364-8782", "clpid": "Fultz-B-T" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "To avoid the challenges of crystallization and the size limitations of NMR, it has long been hoped that single-particle cryo-electron microscopy (cryo-EM) would eventually yield atomically interpretable reconstructions. For the most favorable class of specimens (large icosahedral viruses), two of the key obstacles are the large computational requirements of high-resolution reconstructions and the curvature of the Ewald sphere, which leads to a breakdown of the projection theorem used by conventional 3D reconstruction programs. Here, two solutions to these obstacles are presented.
\r\n\r\nFirst, a simple distributed processing system named Peach was developed to meet the rising computational demands of modern structural biology (and other) laboratories without additional expense by using existing hardware resources more efficiently. A central server distributes jobs to idle workstations in such a way that each computer is used maximally, but without disturbing intermittent interactive users. As compared to other distributed systems, Peach is simple, easy to install, easy to administer, easy to use, scalable, and robust. While it was designed to queue and distribute large numbers of small tasks to participating computers, it can also be used to send single jobs automatically to the fastest currently available computer and/or survey the activity of an entire laboratory's computers. Tests of robustness and scalability are reported, as are three specific cryo-EM applications where Peach enabled projects that would not otherwise have been feasible without an expensive, dedicated cluster.
\r\n\r\nSecond, an iterative refinement reconstruction algorithm, Prec, is described that overcomes the curvature of the Ewald sphere resolution limitation by averaging information from images recorded from different points of view, as are present in typical micrographs. Prec was implemented in the popular software packages IMIRS, EMAN, and Bsoft. In preliminary tests with both simple and multi-slice simulated images, Prec overcame the curvature problem even in the presence of noise. Prec was then used to refine the three recently published, ~ 4 \u00c5 resolution, icosahedral virus reconstructions from experimental cryo-EM images, but unfortunately no significant improvements in resolution were realized. Further simulations showed that limitations other than the Ewald sphere curvature problem must still be dominant in these experimental studies.
\r\n", "doi": "10.7907/GJKS-2P80", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2175", "collection": "thesis", "collection_id": "2175", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272009-091024", "primary_object_url": { "basename": "HK_Privett_thesis.pdf", "content": "final", "filesize": 19168490, "license": "other", "mime_type": "application/pdf", "url": "/2175/10/HK_Privett_thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "An Iterative Approach to de novo Computational Enzyme Design and the Successful Application to the Kemp Elimination", "author": [ { "family_name": "Privett", "given_name": "Heidi Kathleen", "clpid": "Privett-Heidi-Kathleen" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The development of reliable methods for the 'on demand\" de novo design of an enzymatic catalyst for an arbitrary chemical reaction has been an elusive goal of the computational protein design community. Recent successful results of de novo computational enzyme design have been encouraging, but the activity of the enzymes produced so far is still well below that of natural enzymes and the generalizability of these methods has yet to be established.
\r\n\r\nPresented in this thesis are methods that we have developed for the computational design of enzyme active sites as well as results from the evaluation of these methods through a test case, the Kemp elimination. Initial Kemp elimination designs were shown to be inactive. However, in the course of refining these design procedures, we carried out extensive theoretical and experimental evaluation of several of these inactive designs, which allowed us to identify the causes of the inactivity and led to adjustments of our design procedure. These modified methods were then successfully used to design four distinct enzymes for this reaction in three inert scaffolds including the scaffold that housed the previously inactive designs. In addition, we demonstrate that molecular dynamics simulations can accurately predict the activity of designed Kemp elimination enzymes and can be used as a reliable prescreening step, allowing us to focus our experimental efforts on designs that are most likely to be active.
\r\n\r\nThe work presented here demonstrates that the cyclic evaluation and redesign of both active and inactive enzymes was instrumental in the identification and resolution of deficiencies in our computational methods and directly resulted in de novo designed enzymes with novel and increased activity.
\r\n", "doi": "10.7907/1402-XZ32", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2181", "collection": "thesis", "collection_id": "2181", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272009-161836", "primary_object_url": { "basename": "Thesis_Claire_Jacobs.pdf", "content": "final", "filesize": 7936312, "license": "other", "mime_type": "application/pdf", "url": "/2181/10/Thesis_Claire_Jacobs.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structural Modifications to DNA-Binding Polyamides for Improved Biological Activity in Cell Culture", "author": [ { "family_name": "Jacobs", "given_name": "Claire Sigrid", "clpid": "Jacobs-Claire-Sigrid" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Polyamides are a class of synthetic small molecules that recognize DNA in a sequence-specific fashion through a network of hydrogen bonds formed with bonding partners in the floor of the minor groove. The binding affinity of polyamides is comparable to that of numerous DNA-binding proteins, and polyamides have been shown to displace DNA-binding proteins. As such, they present a powerful opportunity to modulate expression levels of genes vital to human health. The cellular permeability and biological activity of polyamides has presented an impediment in moving from in vitro to in vivo work that was partially removed by the discovery that fluorescein dyes facilitate cell entry. The work described here details recent advances in modifications to the C-terminal polyamide linker, linkage and tail groups that improve the endogenous inducible gene regulation activity of polyamides in cell culture.\r\n", "doi": "10.7907/HMMN-YW83", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1685", "collection": "thesis", "collection_id": "1685", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05082009-170032", "primary_object_url": { "basename": "CompleteThesis.pdf", "content": "final", "filesize": 67703519, "license": "other", "mime_type": "application/pdf", "url": "/1685/14/CompleteThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Ultrafast Electron Crystallography: Principles and Applications", "author": [ { "family_name": "Yang", "given_name": "Ding-Shyue (Jerry)", "orcid": "0000-0003-2713-9128", "clpid": "Yang-Ding-Shyue-Jerry" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "thesis_committee": [ { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Yeh", "given_name": "Nai-Chang", "clpid": "Yeh-Nai-Chang" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "During the last 20 to 30 years, the development and application of time-resolved experimental techniques with a femtosecond temporal resolution have brought to us much knowledge about the fundamental processes in physics, chemistry and biology. Nevertheless, standard spectroscopic methods have their limitation in the determination of the transient structures during ultrafast dynamics at the atomic level, because the spatial resolution is restricted by the wavelength of the probe pulse used. In contract, with the scheme of femtosecond optical initiation and electron probing and through the diffraction phenomenon, ultrafast electron crystallography (UEC) was recently developed as a time-resolved structure-probing technique for condensed-matter studies. The short wavelength and small pulse duration of the highly accelerated electrons used provide the atomic-scale spatiotemporal resolution. In addition, the large electron\u2013matter interaction enables the detection of small transient changes as well as the investigation of surface and interfacial phenomena.
\r\n\r\nThis thesis describes the principles of UEC and its applications to a variety of systems, ranging from nanometer-scale structures to highly correlated materials and to interfacial assemblies. By using a prototype semiconducting material, we elucidated the fundamental processes at work in different parts of the femtosecond-to-nanosecond time range; this investigation led to a conceptual change from the consideration of laser-induced heating to the examination of nonequilibrium structural modifications as a result of the transient dynamical changes in, e.g., carriers, the crystal potential, and phonons. On the basis of such an understanding, we observed and understood the colossal unidirectional expansion induced by the photoexcitation of nanostructures to be a potential-driven result rather than a thermal one.
\r\n\r\nFor highly correlated materials, we showed the effectiveness of UEC in resolving the transient intermediate structures during phase transformations as well as identifying new phases in the nonequilibrium state. An important breakthrough made by UEC was the confirmation of the anisotropic involvement of lattice in the electron pairing mechanism for high-temperature superconductors. In interfacial assemblies, we also found a nonequilibrium phase transformation in water and the phenomenon of ultrafast annealing for a better order in a self-assembled monolayer. With these successful experiences, we expect more condensed-matter studies by UEC to come.
\r\n", "doi": "10.7907/Y61P-2B24", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4645", "collection": "thesis", "collection_id": "4645", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-11232008-171327", "primary_object_url": { "basename": "00CompleteThesis.pdf", "content": "final", "filesize": 7590682, "license": "other", "mime_type": "application/pdf", "url": "/4645/1/00CompleteThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Programming Protein Patterns on DNA Nanostructures with Pyrrole-Imidazole Polyamides", "author": [ { "family_name": "Cohen", "given_name": "Justin Delgado", "clpid": "Cohen-Justin-Delgado" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Winfree", "given_name": "Erik", "clpid": "Winfree-E" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Molecular recognition of DNA has important applications for gene regulation, molecular biology, and DNA nanotechnology. Pyrrole-imidazole polyamides are a unique class of molecules with the ability to bind to DNA in a programmable manner. These small molecule analogues of distamycin A can be programmed to target virtually any DNA sequence with high affinity and specificity. Originally characterized for their ability to bind to B-form DNA, polyamides are also able to target DNA in architectures such as the nucleosome core particle (NCP) and two-dimensional DNA nanostructures including DX-arrays and DNA origami. In addressing DNA nanostructures, polyamide-biotin conjugates can be used to create nanoscale molecular assemblies in a bottom-up approach to self-assembly. The ability to address unique sequences on a DNA nanostructure with different polyamides makes it possible to create unique arrangements of protein on a single 2-dimensional DNA template. Polyamides targeted to the NCP can be used for a variety of exciting applications including NCP-templated ligation reactions, gene regulation, and as tools for X-ray crystallography. The programmability of polyamides makes them an ideal tool for addressing a variety of DNA architectures for varying applications.\r\n", "doi": "10.7907/CZ2K-MJ03", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4147", "collection": "thesis", "collection_id": "4147", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10172008-222221", "primary_object_url": { "basename": "Anderson_Thesis.pdf", "content": "final", "filesize": 47347332, "license": "other", "mime_type": "application/pdf", "url": "/4147/1/Anderson_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Microfluidics-Based Strategies for Protein Crystallography", "author": [ { "family_name": "Anderson", "given_name": "Megan Jo", "clpid": "Anderson-Megan-Jo" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Protein crystallography is an invaluable tool for the study of biological processes at the molecular level. While several crystallization techniques are actively pursued in both academic and industrial laboratories to produce high-quality protein crystals, the use of microfluidic technology for structural biology was previously shown to improve protein crystallization over more traditional methods. This thesis describes a microfluidics-based crystallization strategy that was developed to increase the success rate of crystallizing challenging proteins. The crystallization strategy involves using multiple microfluidic devices to characterize the solubility trends of the crystallization target, to perform nanoliter volume free interface diffusion crystallization experiments designed around the solubility trends, and to enable in situ diffraction analysis of crystals grown in microfluidic devices. The crystallization strategy was applied to the crystallization of a dozen challenging proteins and increased the overall crystallization and diffraction success rates compared with conventional automation. The crystallization strategy was also utilized to crystallize four metabolic proteins and provides the first demonstration of in situ structure determination for novel crystallization targets using a microfluidic crystallization platform. Additional technological advances were accomplished by the development of a novel microfluidic device designed to address the specific challenges of membrane protein crystallography. To date, this microfluidic crystallization strategy has produced four novel protein structures and holds great promise for future work in the field of protein crystallography.\r\n", "doi": "10.7907/P5ZD-0S21", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2485", "collection": "thesis", "collection_id": "2485", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062009-131454", "primary_object_url": { "basename": "Ursell_Thesis_2009.pdf", "content": "final", "filesize": 26146411, "license": "other", "mime_type": "application/pdf", "url": "/2485/1/Ursell_Thesis_2009.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stretching the Definition of a Lipid Bilayer: Elasticity's Role in Protein and Lipid Organization", "author": [ { "family_name": "Ursell", "given_name": "Tristan Scott", "orcid": "0000-0001-9273-8413", "clpid": "Ursell-Tristan-Scott" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Klug", "given_name": "William", "clpid": "Klug-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Huang", "given_name": "Kerwyn", "clpid": "Huang-Kerwyn" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The Central Dogma forms the foundation of molecular biology couched in polymer language; all the key players are there \u2014 DNA, RNA, protein \u2014 or so it would seem. Yet one class of biologically synthesized molecules, crucial for life, is often over looked: lipids. These amphiphilic molecules exhibit a number of strange properties, integral to the cells ability to separate self from non-self in a chemically diverse environment. Lipids self-assemble into two-dimensional bi-layered fluids with aspect ratios of a thousand to one or more, capable of self-healing and bending into extraordinarily complex shapes. Within the cell, membranes allow for numerous chemically-distinct compartments, essential for metabolism, protein assembly, genome management, and cell division. With literally hundreds of different kinds of lipids and proteins interacting on a given membrane, we have much to learn about how membranes regulate the flow of materials into and out of cells. Clearly, molecular level detail is integral to our understanding of these systems, however, on the mesoscopic level membranes exhibit certain mechanical effects that serve to organize lipids and proteins, the study of which forms the bulk of this dissertation. We start by building an elastic model of bilayers, where embedded proteins deform the surrounding membrane and incur a free energy cost. This allows the mechanical attributes of the bilayer to influence the conformation of embedded proteins. We explore this connection in the context of mechanosensation in bacteria, as well as developing methods that allow bilayer mechanics to comment on the structure of classically voltage-gated ion channels. In addition to affecting conformational preferences, these same deformations have a finite length-scale that results in interactions between embedded proteins. Depending on the protein shape, these interactions can be attractive or repulsive, may exert torques on proteins, provide for a mechanism of shape-specific oligomerization, and importantly allow proteins to utilize the bilayer as a generic communicator of conformational information. The effects of these elastic interactions are discussed in the context of mean protein spacing, dimerization, conformational cooperativity, and likely pathways to multi-mer protein assembly, with the bacterial mechanosensitive channel MscL as a structural example. In subsequent chapters, bilayer elasticity is used to shed light on the large-scale organization of lipids themselves. Biological membranes likely have multiple fluid, lipid phases, where sequestration of saturated lipids and cholesterol form lipid domains. We found that formation of domains above a certain critical size induces morphological transitions to a \u2018dimpled\u2019 phase which turns on repulsive, elastic interactions that serve to spatially organize domains as well as severely inhibit domain coalescence. This provides a mechanism for the maintenance of lipid lateral heterogeneity on relatively short length-scales and long time scales. We further observed discrete transitions to a \u2018budded\u2019 domain morphology and developed a set of interpretive energetic transition rules between flat, dimpled and budded domains. We demonstrate that these morphologies and their attendant transitions lead to a unique form of domain-size-dependent transport in membranes. Further, we employ the mechanics of vesicles to model osmoregulation via channel proteins, and in the setting of conserved surface area and volume to develop a theoretical and experimental framework to study membrane adhesion in the context of the homophilic protein binding.\r\n", "doi": "10.7907/Q0R5-K353", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2485", "collection": "thesis", "collection_id": "2485", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062009-131454", "primary_object_url": { "basename": "Ursell_Thesis_2009.pdf", "content": "final", "filesize": 26146411, "license": "other", "mime_type": "application/pdf", "url": "/2485/1/Ursell_Thesis_2009.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stretching the Definition of a Lipid Bilayer: Elasticity's Role in Protein and Lipid Organization", "author": [ { "family_name": "Ursell", "given_name": "Tristan Scott", "orcid": "0000-0001-9273-8413", "clpid": "Ursell-Tristan-Scott" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Roukes", "given_name": "Michael Lee", "orcid": "0000-0002-2916-6026", "clpid": "Roukes-M-L" }, { "family_name": "Klug", "given_name": "William", "clpid": "Klug-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Huang", "given_name": "Kerwyn", "clpid": "Huang-Kerwyn" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "The Central Dogma forms the foundation of molecular biology couched in polymer language; all the key players are there \u2014 DNA, RNA, protein \u2014 or so it would seem. Yet one class of biologically synthesized molecules, crucial for life, is often over looked: lipids. These amphiphilic molecules exhibit a number of strange properties, integral to the cells ability to separate self from non-self in a chemically diverse environment. Lipids self-assemble into two-dimensional bi-layered fluids with aspect ratios of a thousand to one or more, capable of self-healing and bending into extraordinarily complex shapes. Within the cell, membranes allow for numerous chemically-distinct compartments, essential for metabolism, protein assembly, genome management, and cell division. With literally hundreds of different kinds of lipids and proteins interacting on a given membrane, we have much to learn about how membranes regulate the flow of materials into and out of cells. Clearly, molecular level detail is integral to our understanding of these systems, however, on the mesoscopic level membranes exhibit certain mechanical effects that serve to organize lipids and proteins, the study of which forms the bulk of this dissertation. We start by building an elastic model of bilayers, where embedded proteins deform the surrounding membrane and incur a free energy cost. This allows the mechanical attributes of the bilayer to influence the conformation of embedded proteins. We explore this connection in the context of mechanosensation in bacteria, as well as developing methods that allow bilayer mechanics to comment on the structure of classically voltage-gated ion channels. In addition to affecting conformational preferences, these same deformations have a finite length-scale that results in interactions between embedded proteins. Depending on the protein shape, these interactions can be attractive or repulsive, may exert torques on proteins, provide for a mechanism of shape-specific oligomerization, and importantly allow proteins to utilize the bilayer as a generic communicator of conformational information. The effects of these elastic interactions are discussed in the context of mean protein spacing, dimerization, conformational cooperativity, and likely pathways to multi-mer protein assembly, with the bacterial mechanosensitive channel MscL as a structural example. In subsequent chapters, bilayer elasticity is used to shed light on the large-scale organization of lipids themselves. Biological membranes likely have multiple fluid, lipid phases, where sequestration of saturated lipids and cholesterol form lipid domains. We found that formation of domains above a certain critical size induces morphological transitions to a \u2018dimpled\u2019 phase which turns on repulsive, elastic interactions that serve to spatially organize domains as well as severely inhibit domain coalescence. This provides a mechanism for the maintenance of lipid lateral heterogeneity on relatively short length-scales and long time scales. We further observed discrete transitions to a \u2018budded\u2019 domain morphology and developed a set of interpretive energetic transition rules between flat, dimpled and budded domains. We demonstrate that these morphologies and their attendant transitions lead to a unique form of domain-size-dependent transport in membranes. Further, we employ the mechanics of vesicles to model osmoregulation via channel proteins, and in the setting of conserved surface area and volume to develop a theoretical and experimental framework to study membrane adhesion in the context of the homophilic protein binding.\r\n", "doi": "10.7907/Q0R5-K353", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1883", "collection": "thesis", "collection_id": "1883", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05192009-163326", "primary_object_url": { "basename": "Adrian_Rice_Full_Thesis.pdf", "content": "final", "filesize": 27472401, "license": "other", "mime_type": "application/pdf", "url": "/1883/1/Adrian_Rice_Full_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Biophysical and Cell Biological Studies Characterizing the Vertebrate Iron Exporter Ferroportin", "author": [ { "family_name": "Rice", "given_name": "Adrian Edward", "clpid": "Rice-Adrian-Edward" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "orcid": "0000-0003-1556-4864", "clpid": "Jensen-G-J" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Mammalian iron homeostasis is maintained by an intricate network of diverse proteins that constantly survey systemic iron levels and carefully regulate the uptake of iron from the diet. Control of this uptake is critically important because once iron is absorbed, mammals have no regulated mechanism for its removal. The portal through which iron enters the body is ferroportin, a multipass membrane protein expressed on the basolateral membrane of epithelial cells in the duodenum. The iron export function of ferroportin is primarily regulated by the serum peptide hormone hepcidin, which is secreted from the liver when systemic iron levels are high. Hepcidin acts as a negative regulator of iron uptake by binding to ferroportin at the cell surface and inducing its internalization and degradation. Genetic defects in ferroportin, hepcidin, or the proteins involved with sensing systemic iron levels lead to iron overload diseases known as hereditary hemochromatosis. Using the tools of biophysics and cell biology, we sought to study ferroportin and its interaction with hepcidin in order to better understand this critical bottleneck in iron uptake and how genetic defects within ferroportin might lead to disease. We developed the first protocols for the overexpression, detergent-solubilization, and purification of recombinant ferroportin. We determined that detergent-solubilized ferroportin is a monomer capable of binding hepcidin in vitro. We characterized the expression and subcellular localization of ferroportin in mammalian tissue culture and determined that both the amino- and carboxy-termini of ferroportin are cytosolic. We developed cell-based assays for the hepcidin-induced internalization of ferroportin and used these to characterize the route of internalization from the plasma membrane through early endosomes to degradative lysosomal compartments. Using live-cell imaging techniques, we showed that this internalization depended on intact microtubules. We expanded this cell-biological study to include sixteen disease-related ferroportin mutants and reported that each mutant was expressed on the plasma membrane like wild-type ferroportin, but that only a subset of the mutants were capable of being internalized by hepcidin. These studies form a foundation for future biophysical and cell-biological studies of ferroportin function. ", "doi": "10.7907/2P16-3X70", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1570", "collection": "thesis", "collection_id": "1570", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05012009-112749", "primary_object_url": { "basename": "BDA-thesis-final.pdf", "content": "final", "filesize": 5847746, "license": "other", "mime_type": "application/pdf", "url": "/1570/10/BDA-thesis-final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Development and Validation of Optimization Methods for the Design of Protein Sequences and Combinatorial Libraries", "author": [ { "family_name": "Allen", "given_name": "Benjamin Daniel", "clpid": "Allen-Benjamin-Daniel" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "To facilitate the design of protein sequences with desired properties, simulation techniques have been developed to allow large portions of amino acid sequence space to be evaluated by computer. These computational protein design methods apply optimization algorithms to sort through the enormity of sequence space and find desirable variants.
\r\n\r\nSimple modifications to the stochastic optimization algorithm FASTER enhanced its performance by two orders of magnitude without loss of accuracy, and rendered it more efficient than its major competitor by a factor of 10. These improvements allowed higher-quality amino acid solutions to be found more quickly, and accelerated the pace at which users could perform cycles of design and model adjustment.
\r\n\r\nThis success prompted research into techniques for a protein design formulation that allows simulation in the context of multiple states simultaneously. This multi-state design can be used to wield explicit control over structural, binding, or catalytic specificity, and changes the scope of design goals that can be addressed by computation. Evaluation of multi-state FASTER indicated that it performed radically better than its major competitor in a variety of design contexts, and that in most cases it found solutions better than those that could ever be found using a lesser method.
\r\n\r\nMulti-state optimization using FASTER was applied to test the influence of various types of input structural data on the design of a small protein. To facilitate this evaluation, methods for the design and high-throughput stability screening of combinatorial libraries were developed. Screening of libraries based on single structures and structural ensembles indicated the success of multi-state modeling. Our results also suggested that the exhaustive screening of designed libraries can help to elucidate the origins of design model failures. Finally, they showed that success of a design procedure does not hinge on its ability to correlate experimental and simulated measures of fitness, and prompted greater consideration of design methods that target explicitly conformational specificity.
\r\n", "doi": "10.7907/SNJJ-0Q88", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1536", "collection": "thesis", "collection_id": "1536", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04282009-211225", "primary_object_url": { "basename": "00CompleteThesis.pdf", "content": "final", "filesize": 21277358, "license": "other", "mime_type": "application/pdf", "url": "/1536/1/00CompleteThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Structural Studies of Cyclic Py-Im Polyamides", "author": [ { "family_name": "Chenoweth", "given_name": "David Michael", "orcid": "0000-0002-0819-4669", "clpid": "Chenoweth-David-Michael" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work presented in this thesis is focused on the molecular recognition of DNA by minor groove binding polyamides. Methods and strategies for the solution-phase synthesis of hairpin and cyclic pyrrole-imidazole polyamides are presented with optimized protocols requiring little to no chromatography. These synthetic strategies have led to the design of cyclic polyamides targeted to the androgen response element and are shown to be biologically active and cell permeable in cell culture experiments in addition their binding affinities rival that of most polyamide architectures. The structural elucidation of an \u03b1-amino-turn-linked cyclic polyamide is presented at 1.17 \u00c5 resolution providing insight into the detailed molecular recognition process and allosteric modulation responsible for the inhibition of transcription factor-DNA binding. Additionally, structural elucidation of a \u03b2-amino-turn-linked cyclic polyamide, highlighting the conformational differences compared to the \u03b1-amino-turn linked structure is presented. A structural basis for the inability of polyamides to bind dsRNA is also proposed based on biophysical, structural, and modeling data. In addition to these studies a new class of programmable oligomers targeting the DNA sequence 5\u2019-WGGGGW-3\u2019 were shown to inhibit DNA binding of the Nf-kB transcription factor by EMSA gel shift. Compounds synthesized in this study were found to possess unique fluorescent properties with the ability to modulate their fluorescence by binding their targeted dsDNA, leading to sequence specific fluorescent detection reagents. Efforts toward the templated-assembly of polyamides using higher-order DNA structure (NCP) are also reported and the development of a new pro-fluorescent class of heterocycle, which has the potential to be used as a chemical reporter of ligation events is described.\r\n", "doi": "10.7907/PZEC-VA33", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2268", "collection": "thesis", "collection_id": "2268", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292009-094239", "primary_object_url": { "basename": "1-FullThesis.pdf", "content": "final", "filesize": 3698531, "license": "other", "mime_type": "application/pdf", "url": "/2268/1/1-FullThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Adrenergic Receptors: Model Systems for Investigation of GPCR Structure and Function", "author": [ { "family_name": "Wiencko", "given_name": "Heather L.", "clpid": "Wiencko-Heather-L" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Membrane proteins mediate intercellular communication, resulting in changes in the membrane and within the cell itself. One superfamily of integral membrane proteins, G-protein coupled receptors (GPCRs), are responsible for a vast diversity of processes. Their conformational flexibility and membrane environment pose challenges for direct structural characterization, and to date only five of the more than 1,000 known GPCRs have been characterized by high-resolution crystallography.
\r\n\r\nThe nine adrenergic GPCRs mediate the stress response throughout the body, and are implicated in diseases including hypertension and asthma. While they are among the best studied families of GPCRs, much remains to be learned about selectivity and activation. The first section of this work describes the ab initio structure prediction of the turkey beta-1 receptor and validation using a series of stabilizing mutations. This work preceded the currently available turkey beta-1 structure but shows good agreement, especially in the binding site. It validates the latest methods developed for GPCR structure prediction, emphasizes the role of a neutral charge scheme in energy determination, and explores a structure validation strategy based on stabilizing mutations rather than ligand docking. The next section uses the experimental beta-1 crystal structure as a starting point for nanosecond timescale molecular dynamics, exploring the roles of ligand binding in helix movement that contribute to the transition to an active state. These simulations reveal the early steps in receptor activation, beginning with tilting motions of transmembrane helices 5 and 6 and movement of transmembrane helix 1 closer into the protein core. The last section presents homology models of the human adrenergic receptors for which there are not yet crystal structures. The receptors most closely related to the target structures show the best results, while the less related ones will require further refinement. The best structures provide insight into the binding site of subtype selective antagonists, and can serve as the foundation for future studies. Over the course of these explorations, new subtleties in adrenergic structure have been illuminated, and may drive further exploration into selective binding and the activation mechanism of these and other receptors.
", "doi": "10.7907/S3RC-RZ59", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:5203", "collection": "thesis", "collection_id": "5203", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262008-115504", "primary_object_url": { "basename": "TDB-thesis_final.pdf", "content": "final", "filesize": 5532126, "license": "other", "mime_type": "application/pdf", "url": "/5203/7/TDB-thesis_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Development of Enantioselective Organocatalytic Technologies for the Alpha-Functionalization of Aldehydes and Ketones", "author": [ { "family_name": "Beeson", "given_name": "Teresa Diane", "clpid": "Beeson-Teresa-Diane" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The development of an expeditious and room-temperature conversion of aliphatic aldehydes to chiral terminal epoxides is described. \u03b1-Chloroaldehydes were prepared via asymmetric enamine catalysis with an imidazolidinone catalyst followed by in situ reduction and cyclization to generate the terminal epoxide. Epoxides with a variety of aliphatic groups and functionalities were produced in 75 minutes with good yields and excellent selectivities.
\r\n\r\nThe catalytic enantioselective direct \u03b1-fluorination of aldehydes and ketones is also reported. \u03b1-Fluoroaldehydes were conveniently prepared via enamine catalysis with an imidazolidinone catalyst and N-fluorobenzenesulfonimide (NFSI) as an electrophilic fluorine source. The method tolerated a wide variety of aldehyde substrates and functional groups. Catalyst loadings as low as 1 mol% generated the fluorinated products in good yield and excellent enantioselectivity. Additionally, various catalyst architectures were studied to apply the \u03b1-fluorination reaction to ketone substrates. Cinchona alkaloid-derived catalysts were found to successfully facilitate the \u03b1-fluorination of ketones in high yields and excellent enantioselectivities.
\r\n\r\nAlso presented is the advent of SOMO catalysis, a new mode of organocatalytic activation based on the catalytic generation of radical cations. A secondary amine catalyst reacts with an aldehyde to transiently generate an enamine that, in turn, undergoes a single-electron oxidation to yield a stabilized radical cation that is subject to enantiofacial discrimination. While the parent enamine reacts only with electrophiles, the radical cation combines with SOMO nucleophiles at the same reacting center, thereby enabling a diverse range of previously unknown asymmetric transformations. As a first example and proof of principle, the development of the direct and enantioselective \u03b1-allylation of aldehydes using SOMO catalysis is described.
\r\n", "doi": "10.7907/YYTK-TP61", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:4998", "collection": "thesis", "collection_id": "4998", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12142007-125144", "primary_object_url": { "basename": "Thesis_XX.pdf", "content": "final", "filesize": 2652120, "license": "other", "mime_type": "application/pdf", "url": "/4998/1/Thesis_XX.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids", "author": [ { "family_name": "Xiu", "given_name": "Xinan Joanne", "clpid": "Xiu-Xinan-Joanne" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nicotinic acetylcholine receptors (nAChR) are an important family of ligand gated ion channels found throughout the CNS and the PNS. They have been indicated in a series of physiological functions and pathological states. nAChRs have received extensive study in the past as a prototype of the Cys loop LGIC member. Growing interest in developing subtype specific agents targeting nAChRs to treat neurological diseases require more detailed structural and functional information in the numerous members of the nAChR family.
\r\n\r\nWe performed structure-function studies on the chemical scale of several of the most important members of this family using a powerful combination of conventional mutagenesis and unnatural amino acid incorporations. Chapter 2 describes our research in studying the channel gating mechanism of the prototypic nAChR, the muscle type (\u03b1\u2081)\u2082\u03b2\u03b3\u03b4. We studied thoroughly the gating interface of the receptor and concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily. Chapter 3 reports our studies in the ligand binding mechanism of the most prevalent neuronal type \u03b14\u03b22 and \u03b17 nAChR. We identified a cation-\u03c0 interaction and a hydrogen bond employed by nicotine with the \u03b14\u03b22 receptor. These two key interactions are absent or significantly diminished in both the muscle type receptors and in the \u03b17 form of neuronal receptor. In Chapter 4 we studied the ligand binding mechanism of a relatively newly characterized neuronal receptor, \u03b14\u03b24.
\r\n\r\nFrom these studies, we found that in the Cys loop superfamily, homology in amino acid sequences and structures do not translate into a shared functional mechanism. In fact, different sets of chemical interactions are adopted between ligands and the receptor, and between amino acids within the ion channel proteins, both in ligand binding and channel gating.
\r\n\r\nIon channels are membrane bound multi-subunit macromolecules. We are able to carry out such exhaustive detailed structure-function studies by means of the fast developing methodology of unnatural amino acid incorporation by nonsense suppression. This thesis also describes our effort to improve the efficiency of nonsense suppression. In particular, we designed multiple 21nt small interfering RNA (siRNA) targeting release factor 1 (eRF1) in both HEK cells and Xenopus oocytes, and monitored the nonsense suppression efficiency change in vivo and in vitro by RNA PCR, Western blotting, fluorescence, and electrophysiology (Chapter 5).
\r\n", "doi": "10.7907/HWGS-7Z13", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:3212", "collection": "thesis", "collection_id": "3212", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08232007-154048", "primary_object_url": { "basename": "Stafford_Thesis_Complete.pdf", "content": "final", "filesize": 47413801, "license": "other", "mime_type": "application/pdf", "url": "/3212/8/Stafford_Thesis_Complete.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Design of Protein-DNA Dimerizers", "author": [ { "family_name": "Stafford", "given_name": "Ryan Leonard", "clpid": "Stafford-Ryan-Leonard" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Genes are regulated by proteins called transcription factors that bind to DNA in a sequence-specific manner and modulate the rate of transcription. Mutated transcription factors often lead to abnormal gene expression, developmental defects, and disease. This thesis describes the design of chemicals called protein-DNA dimerizers that mimic natural transcription factor protein-DNA complexes. In the long-term, it is hoped that these dimerizers will be able to engage or even replace mutant transcription factors and artificially regulate gene expression in living cells. Specifically, programmable DNA binding pyrrole-imidazole polyamides conjugated to YPWM peptide motifs incorporating various linker domains facilitate the binding of a natural transcription factor, extradenticle, to DNA. From a design point of view, it has been explored what the minimum size and shape (branched or linear) is that will ultimately be optimal for cell uptake with adequate functional potency in the transcriptional apparatus. Branched dimerizers are shown to function with a minimal WM dipeptide protein-binding domain in vitro up to 37 degrees C, and linear dimerizers are shown to function with WMK tripeptides up to 20 degrees C. Collectively, branched and linear dimerizers can facilitate protein binding to DNA from 2 base pair overlap sites to ones that reach 6 base pairs apart. Polyamide-WM-fluorescein conjugates are also found to be cell permeable in several cell lines including HeLa, MCF-7, and PC3. These studies provide insight into the importance of linker length and composition, binding-site spacing and orientation, and the protein binding domain content that are important for the optimization of protein DNA-dimerizers suitable for biological experiments.", "doi": "10.7907/PF5M-KF68", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2323", "collection": "thesis", "collection_id": "2323", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302008-164022", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 5547848, "license": "other", "mime_type": "application/pdf", "url": "/2323/1/Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Methods in Computational Protein Design", "author": [ { "family_name": "Kam", "given_name": "Victor Wai Tak", "clpid": "Kam-Victor-Wai-Tak" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In silico design of protein has generated enormous interest with the rapid advances in computational power. Biological systems are known for their complexity, and we have made a series of computational developments that allow us to perform computational protein design. In this work we present a methodology for the design and prediction of protein active sites.
\r\n\r\nWe begin by presenting SCREAM, a program developed to accurately position sidechains in proteins. We show how using an improved scoring function and placement algorithm allow us to achieve better accuracy in the placement and prediction of sidechains in proteins compared to other methods.
\r\n\r\nWe then describe the development of an accurate treatment for describing hydrogen bonding. This is done by refining the hydrogen bond term in the force field DREIDING. We also need to properly describe electrostatics effects in proteins, and to this end, we introduce neutralized residues for proteins. We found that this improves the variance in our predictions dramatically.
\r\n\r\nFinally, having established the components described above, we describe a protein design methodology encompassing the above methods and tools. We show predictions we made and those having been verified by experiments.
\r\n", "doi": "10.7907/S7KW-0M44", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:1806", "collection": "thesis", "collection_id": "1806", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05142008-113003", "primary_object_url": { "basename": "Seitaridou_thesis.pdf", "content": "final", "filesize": 56672776, "license": "other", "mime_type": "application/pdf", "url": "/1806/1/Seitaridou_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Non-Equilibrium Dynamics: Diffusion in Small Numbers and Ribosomal Self-Assembly", "author": [ { "family_name": "Seitaridou", "given_name": "Effrosyni", "orcid": "0000-0002-1668-6786", "clpid": "Seitaridou-Effrosyni" } ], "thesis_advisor": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "thesis_committee": [ { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bockrath", "given_name": "Marc William", "clpid": "Bockrath-M-W" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Biological systems are encountered in states that are far from equilibrium. A change in the cell's condition triggers the flow of energy and matter that causes the cell's transition from that non-equilibrium state to a different state. Our interest is on non-equilibrium systems and the way these relate to the cell's \"small numbers\" limit as well as to the mechanisms of self-assembly.
\r\n\r\nCells contain proteins and nucleotides in numbers smaller than Avogadro's. In addition, advances in single-molecule experiments, which are, by definition, a case of the \"small numbers\" problem, have emphasized the importance of fluctuations. Does the result we get from a single-molecule measurement agree with what we would get from a bulk measurement? Is it a fluctuation from the mean? It is, thus, of biological interest to see the behavior of non-equilibrium systems at the \"small numbers\" limit where fluctuations become important. Using microfluidics, we concentrate on the diffusion of a small number of submicron particles in a system that is away from equilibrium. Therefore, we study the \"small numbers\" limit of Fick's Law, with special reference to the fluctuations that attend diffusive dynamics in order to experimentally test the theoretical predictions obtained via the use of E. T. Jaynes' \"principle of maximum caliber.\"
\r\n\r\nThe process of macromolecular self-assembly is also highly dynamical. The system's components come together, defeating in this way entropic effects, to form the system. In the case of the ribosome, whose importance lies in its ability to synthesize proteins, understanding the mechanism of the highly dimensional process of self-assembly becomes relevant when designing, for example, new antibiotics. The second part of this thesis concentrates on the RNA-protein interactions which, in the case of the ribosome, determine the mechanism of self-assembly. With the use of microfluidic technology and a fluorescence assay we determine the thermodynamics and kinetics of RNA folding and RNA-protein binding for a fragment of the bacterial 30S ribosomal subunit, paving the way for the study of the complete assembly of the 30S subunit.
\r\n", "doi": "10.7907/X12R-DA46", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:1640", "collection": "thesis", "collection_id": "1640", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05052008-121818", "primary_object_url": { "basename": "Thesis_Final.pdf", "content": "final", "filesize": 4542844, "license": "other", "mime_type": "application/pdf", "url": "/1640/1/Thesis_Final.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structural Studies of the E. coli Methionine ABC Transporter and Its Cognate Binding Protein", "author": [ { "family_name": "Kadaba", "given_name": "Neena Sujata", "clpid": "Kadaba-Neena-Sujata" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "ATP binding cassette (ABC) transporters use ATP hydrolysis to facilitate the transfer of diverse substrates across the membrane. Members of the methionine uptake transporter family, thought to be of considerable biological interest, have not been structurally characterized thus far. The crystal structure of the methionine importer MetNI from Escherichia coli has been solved to 3.7 \u00c5 resolution. The inward-facing conformation of this transporter adopts a more extreme arrangement than seen previously. While the permease domain consists of just five transmembrane helices per monomer, the ATP-binding cassette domain possesses a C-terminal domain in addition to the conserved architecture shared amongst this family. Analysis of the C-terminal extension has revealed a regulatory domain found in other proteins involved in amino acid metabolism, and further classifies this protein as part of the ACT family. Methionine binding in this region suggests a novel mechanism for regulation of transport that possibly stabilizes the inactive conformation of this family of transporters, as this domain is positioned between the nucleotide binding domains. Additionally, crystallization studies of the cognate binding protein to the MetNI system, MetQ were successful. The structure of the MetQ binding protein from E. coli was solved to 1.8 \u00c5 resolution, revealing a bi-lobed structure consistent with many other substrate binding proteins, yet possessing a few differences when compared with previously characterized methionine binding proteins from other organisms. The substrate binding pocket revealed a bound L-methionine residue, which shares key features with other methionine binding proteins and appears to be appropriately selective for L-methionine binding. These combined studies have provided insight into the methionine uptake system and into the ABC transporter mechanism of transport. \r\n", "doi": "10.7907/3DRS-F441", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:1654", "collection": "thesis", "collection_id": "1654", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05062008-171457", "primary_object_url": { "basename": "00thesis.pdf", "content": "final", "filesize": 4852909, "license": "other", "mime_type": "application/pdf", "url": "/1654/1/00thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Characterizing \u03b1-Synuclein Membrane Bound Structure", "author": [ { "family_name": "Lai", "given_name": "Bert Tsunyin", "clpid": "Lai-Bert-Tsunyin" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A feature of Parkinson's disease is the presence of fibrillar protein deposits composed mostly of \u03b1-synuclein and calcium ions in the brain\u2019s substantia nigra region. Although \u03b1-synuclein is natively unfolded, the N-terminal region of the protein is highly helical in the presence of membrane mimics, such as acidic phospholipid vesicles and SDS micelles. The C-terminal region of \u03b1-synuclein is known to bind to calcium ions and modulates aggregation. In this thesis, the structure of \u03b1-synuclein variants, incorporated with tryptophan and 3-nitrotyrosine as donor and energy acceptor pairs, have been characterized in the presence of SDS micelles, small unilammelar vesicles, and calcium ions by various techniques. Distance distributions extracted from time-resolved fluorescence energy-transfer measurements provide site-specific information on the protein conformations. In addition, similar studies using mutants linked to early onset Parkinson\u2019s disease were also performed to investigate the structural effect caused by these mutations. Furthermore, single tryptophan mutants have been designed as fluorescent reporters. The locations of these different tryptophan residues in the bilayer were probed by lipids labeled with bromine and dinitrophenol quenchers. Finally, preliminary studies of the intramolecular structure of \u03b1-synuclein aggregates have been carried out, while elucidation of intermolecular \u03b1-synuclein aggregate structures was made possible by the synthesis of new dyes that allow for long-range fluorescent energy transfer.\r\n", "doi": "10.7907/ZWH6-3B84", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2259", "collection": "thesis", "collection_id": "2259", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292008-140215", "primary_object_url": { "basename": "clv_thesis.pdf", "content": "final", "filesize": 14756797, "license": "other", "mime_type": "application/pdf", "url": "/2259/1/clv_thesis.pdf", "version": "v1.0.0" }, "type": "thesis", "title": "Development and Evaluation of Protein Design Methods for Functional Targets", "author": [ { "family_name": "Vizcarra", "given_name": "Christina Luisa", "clpid": "Vizcarra-Christina-Luisa" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Computational protein design seeks to identify amino acid sequences that will fold into a specified three-dimensional structure. Extending this technique from identification of sequences that retain a native structure to the design of sequences that will carry out a function has been a significant challenge. Modeling the energetics of catalysis and binding requires considerations that may not be necessary for the design of folded, stable proteins. I have investigated models for protein electrostatics with the goal of improving current methods for the design of functional molecules. The work in this thesis is focused on the Poisson-Boltzmann model, a dielectric continuum model that describes the effect of solvent polarization on the electrostatic potential in a protein. I found that this model is amenable to design calculations, as judged by its ability to be decomposed into terms that are used in sequence selection.
\r\n\r\nAside from energy estimation, there are a number of assumptions that are made in protein design in order to make the problem computationally tractable. Because of these assumptions, and also because of incomplete models of protein function, it is expected that many proteins sequences will need to be experimentally characterized to find one that meets a difficult design goal. To this end, I examined methods for using computational tools to produce libraries of protein sequences. These studies showed that (1) structure-based, computational library design methods can be used to generate libraries with a high number of folded proteins and (2) computational design is a promising tool for generating highly mutated proteins with a diverse range of functions.
", "doi": "10.7907/G16E-JZ97", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2262", "collection": "thesis", "collection_id": "2262", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292008-144531", "primary_object_url": { "basename": "Thesis_Entire.pdf", "content": "final", "filesize": 26042748, "license": "other", "mime_type": "application/pdf", "url": "/2262/1/Thesis_Entire.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Characterization and Structure of Mechanosensitive Channels of Small Conductance", "author": [ { "family_name": "Poon", "given_name": "Yan Shuen", "clpid": "Poon-Yan-Shuen" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes the investigation of the mechanosensitive channel of small conductance (MscS). The Escherichia coli MscS structure shows a homoheptameric ion channel with each monomer consisting of 3 transmembrane (TM) helices leading into the cytoplasmic domain. TM3 from each of the 7 subunits form the pore that opens up into a cytoplasmic cage, thought to be a molecular sieve. In order to further knowledge regarding the mechanism behind bacterial mechanosensation, several approaches were employed. Homologs from organisms indigenous to environments with temperatures ranging from 30\u00b0C to 95\u00b0C were cloned, expressed, and characterized. Chimeras were constructed between MscS homologs from 6 hyperthermophilic organisms and E. coli MscS, exchanging the transmembrane and cytoplasmic domains. Each protein is engineered with an N-terminal His6 tag and a C-terminal FLAG epitope. 4 homologs were found to be cytotoxic while 3 chimeras failed to rescue the osmotic-shock sensitivity of an E. coli mutant strain lacking endogenous mechanosensitive ion channel activity.
\r\n\r\nStudies revolving around the various homologs and chimeras led to the crystallization of the Helicobacter pylori MscS homolog. H. pylori MscS shares approximately 35% in sequence identity and was crystallized in space group P212121 with cell dimensions a = 123.0 A, b = 147.4 A, c = 179.4 A. Diffraction data was collected at the Stanford Synchrotron Radiation Laboratory at beamline 12-2 to 4.75 A resolution. A molecular replacement solution was achieved using the program PHASER with the E. coli MscS model (PDB: 2OAU). The refined structure shows a similar overall topology with E. coli MscS demonstrating a homoheptameric channel. The cytoplasmic domain maintains the same structure while TM1 and TM2 display slightly tighter packing with the pore. The structure of the pore suggests that H. pylori and E. coli MscS are crystallized in the same state.
\r\n", "doi": "10.7907/EBZ2-5D39", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2554", "collection": "thesis", "collection_id": "2554", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112008-094638", "primary_object_url": { "basename": "Otey_FinalThesis.pdf", "content": "final", "filesize": 7056815, "license": "other", "mime_type": "application/pdf", "url": "/2554/1/Otey_FinalThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structural and Functional Exploration of an Artificial Family of Cytochromes P450", "author": [ { "family_name": "Otey", "given_name": "Christopher Richard", "clpid": "Otey-Christopher-Richard" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein families are comprised of numerous sequences that adopt a similar three-dimensional structure and functional properties. The superfamily of cytochromes P450 are an excellent example of a common structural scaffold being utilized for a variety of biological functions. This functional diversity is achieved in Nature through millions of years of evolution to create new and diverse sequences. We have used site-directed recombination guided by the computation algorithm SCHEMA to create an artificial family of cytochromes P450 in the laboratory. Members of this family possess unique properties such as altered activity profiles, increased thermostability and the ability to accept new substrates.
\r\n\r\nWe developed screening tools for the rapid analysis of hundreds of individual P450s. These high-throughput assays include the 4-aminoantipyrine (4-AAP) assay which is capable of detecting the hydroxylation of an aromatic ring. High-throughput carbon monoxide binding facilitates the rapid detection of P450s that correctly incorporate a heme cofactor and are thus properly folded and potentially functional. Finally, a substrate binding assay which measures a spectral shift that occurs when a substrate binds in a P450 active site is described.
\r\n\r\nFourteen double-crossover chimeras created from the bacterial P450s CYP102A1 and CYP102A2 were constructed to calibrate the P450 scaffold for SCHEMA, a computational algorithm used to minimize structural disruption in chimeric proteins. We found that only chimeras with high levels of structural disruption as measured by SCHEMA were unfolded. Among the fourteen chimeras we also observed three different activity profiles based on peroxygenase kinetic assays with the substrates p-nitrophenoxydodecanoic acid (12-pNCA), 2-phenoxyethanol and allyloxybenzene.
\r\n\r\nWe applied this calibration to create an artificial family comprising ~3,000 chimeric heme P450 proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, was analyzed using logistic regression analysis (LRA) to identify key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.
\r\n\r\nThirty-four members of this artificial family were assayed for functional diversity on a set of eight substrates. P450 chimeras were able to exceed the parents in total activity on all eight substrates and were grouped into five different groups based on activity profiles using K-means clustering. Activity profiles on eight substrates were then performed in high throughput to produce a data set of 330 chimeras. The mean percent standard deviation of the activity assays showed the reproducibility of these high-throughput data and further analysis may reveal information about sequence-structure-function relationships.
\r\n\r\nThe products of the catalytic reactions of four chimeric P450s with substrates of human P450s, some of which are drug compounds, were analyzed by HPLC in order to determine their identity. Chimeras were able to produce authentic human metabolites of chlorzoxazone, zoxazolamine and propranolol, showed peroxidase acitivty on 4-aminobiphenyl and produced an unknown product with tolbutamide. Finally, the peroxygenase activity of a mutant P450 heme domain is able to be further altered and enhanced using directed evolution. After two rounds of directed evolution and screening with the 4-AAP assay, we found mutants with altered substrate specificities and an overall enhancement of activity.
\r\n\r\nThe design and high-throughput methodologies described here can be used to create artificial protein families and to discover new and useful protein sequences. Like natural protein families, artificial protein families can be used to identify regions of protein sequence and structure that are important for folding and function. This is especially useful for analyzing protein families with few members or for validating tools for structure prediction and for protein sequence-structure-function analysis. Artificial protein families are also rich in sequence diversity and can provide sources of novel protein function. Using the high-throughput methodologies described here, chimeric P450s with enhanced activity, altered activity profiles, and the ability to hydroxylate drug-like compounds to produce authentic human metabolites were discovered in our artificial family of P450s. These methodologies will hopefully be extended to the study of other protein families and to the creation and discovery of increasingly valuable protein catalysts.
", "doi": "10.7907/MZQM-N011", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5218", "collection": "thesis", "collection_id": "5218", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302008-150414", "primary_object_url": { "basename": "Sarina_Thesis_Full_Final.pdf", "content": "final", "filesize": 2632459, "license": "other", "mime_type": "application/pdf", "url": "/5218/8/Sarina_Thesis_Full_Final.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Akt Phosphorylation of Drosophila Heat-Shock Factor: A Signature for Stress Resistance", "author": [ { "family_name": "Mohanty", "given_name": "Sarina", "clpid": "Mohanty-Sarina" } ], "thesis_advisor": [ { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Dunphy", "given_name": "William G.", "clpid": "Dunphy-W-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The heat-shock response is vital to cellular homeostasis. Drosophila melanogaster heat-shock factor (dHSF) is the primary transcriptional activator in the stress response pathway for induction of heat-shock-mediated gene transcription. This work investigates the potential for dHSF to undergo post-translational modification by phosphorylation and lysine tagging, specifically, direct phosphorylation by kinases and covalent-lysine tagging by ubiquitin, acetyl, and SUMO groups. Direct phosphorylation of, and binding to, dHSF was demonstrated by Akt/PKB kinase. Knock-down of this kinase by RNAi resulted in a heat-shock phenotype for dHSF and the acquired DNA-binding ability characteristic of activated transcription factor. Site-directed mutagenesis of lysines within a putative nuclear localization sequence (NLS) revealed two potential sites for regulation of dHSF activation by post-translational modification. The functional consequences of synergistic Akt phosphorylation and lysine modifications are discussed \u2013 this work implicates a role for direct kinase phosphorylation in regulating the stability of dHSF.", "doi": "10.7907/Z9CV4FQJ", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:4105", "collection": "thesis", "collection_id": "4105", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10152007-175458", "primary_object_url": { "basename": "Beginning.pdf", "content": "final", "filesize": 161532, "license": "other", "mime_type": "application/pdf", "url": "/4105/2/Beginning.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Alpha-Diaminobutyric Acid-Linked Hairpin Polyamide-Alklylator Conjugates ", "author": [ { "family_name": "Tsai", "given_name": "Sherry Mon-Yue", "clpid": "Tsai-Sherry-Mon-Yue" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ability to control gene expression through the use of DNA sequence-specific, cell-permeable molecules holds therapeutic promise. Pyrrole-imidazole polyamides are a class of synthetic ligands that can be programmed to bind a broad repertoire of DNA sequences with affinities and specificities comparable to natural DNA-binding proteins. These ligands are generally linked via a turn moiety, resulting in a \u2018hairpin\u2019 structure. Conjugation of polyamides to the non-specific DNA alkylator chlorambucil produces molecules capable of the sequence-specific alkylation of DNA that can arrest gene transcription. We have identified \u03b1-diaminobutyric acid (\u03b1-DABA) as a new turn moiety that can give polyamide-chlorambucil conjugates distinctive biological properties in cellular and small animal models; this may be due to their increased DNA alkylation specificities relative to the standard \u03b3-DABA-linked conjugates. A general characterization of \u03b1-DABA-linked polyamides and their conjugates is reported.
\r\n\r\nAlso described is the development of a modular synthesis of chondroitin sulfate (CS) glycosaminoglycans \u2014 a class of linear, sulfated oligosaccharides that play critical roles in neuronal development, cell division, and spinal cord injury. CS structure in vivo is complex and heterogeneous, hampering efforts to understand its precise biological roles. Access to CS molecules of precisely defined structures is critical to understanding their structure-function relationships. The reported synthetic route is capable of accessing CS structures of defined lengths and sulfation motifs, providing a new approach to understanding these important molecules.
\r\n", "doi": "10.7907/3S8Y-YD79", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2913", "collection": "thesis", "collection_id": "2913", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07172007-124916", "primary_object_url": { "basename": "Traub_M_thesis_2008.pdf", "content": "final", "filesize": 9204440, "license": "other", "mime_type": "application/pdf", "url": "/2913/1/Traub_M_thesis_2008.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Chemical Functionalization and Electronic Passivation of Gallium Arsenide Surfaces", "author": [ { "family_name": "Traub", "given_name": "Matthew C.", "clpid": "Traub-Matthew-C" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Atwater", "given_name": "Harry Albert", "clpid": "Atwater-H-A" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chemically controlled, low defect-density surfaces are essential for the incorporation of gallium arsenide into solar conversion and optoelectronic devices. Detailed X-ray photoelectron spectroscopic (XPS) studies have been conducted on chemically functionalized GaAs(111)A surfaces. Quantitative analysis of this surface after HCl(aq) etching reveals that it is completely free of observable oxide and As(0) contaminants, and is terminated with nearly a full monolayer of Cl. These surface Ga-Cl bonds have been reacted with the phosphine reagents PCl3 and PEt3, both of which introduce P atoms onto the surface. Direct reaction of PCl3 with the oxide-terminated surface leads to surfaces that are nearly oxide free but contain measurable amounts of As(0). Steady-state photoluminescence (PL) intensity measurements were used to evaluate the effectiveness of these techniques at passivating surface carrier recombination. Consistent with the chemical observations, etched and functionalized surfaces showed enhanced PL, while surfaces functionalized directly with PCl3 did not.\r\n\r\nThe effects of surface functionalization were explored on GaAs nanocrystals chemically synthsized with an oxide capping layer. Transmission electron microscopy and powder X-ray diffraction demonstrated that the particles were anisotropically etched by treatment with HCl(aq). XPS measurements showed that the Cl-terminated particles were almost entirely free of oxide but contained significant As(0) contamination. Further functionalization of the particles with N2H4 or NaSH replaced surface Cl atoms with N or S moieties but did not remove this As(0). The corresponding band gap PL of these particles was quite weak. Annealing the functionalized particles lead to the disappearance of the As(0) and strong enhancement of the PL intensity. These results imply that surface As(0) is a dominant carrier trap on nanoscale GaAs surfaces and should be broadly applicable for improving the performance of GaAs nanocrystals and nanowires.\r\n\r\nFinally, Fermi\u2019s golden rule has been used to develop relationships between rate constants for electron transfer in donor-bridge-acceptor and electrode-bridge-acceptor systems and resistances across metal-bridge-electrode and metal-bridge-tip junctions. This formulation was used to predict resistances for alkanethiolate, oligophenylene, and DNA bridges from reported donor-acceptor electron-transfer measurements in these systems. These predicted values were compared to reported resistances measured for these molecules.", "doi": "10.7907/2A4E-HV45", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2245", "collection": "thesis", "collection_id": "2245", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292007-061922", "primary_object_url": { "basename": "14_PRE_CHAPTERS.pdf", "content": "final", "filesize": 214398, "license": "other", "mime_type": "application/pdf", "url": "/2245/14/14_PRE_CHAPTERS.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Refolding a \u03b2-Barrel Membrane Protein\r \r ", "author": [ { "family_name": "Arjara", "given_name": "Gitrada", "clpid": "Arjara-Gitrada" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a \u03b2-barrel membrane protein. The amphiphilic, \u03b2-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus.
\r\n\r\nWhile the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate \u03b2-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate.
\r\n\r\nFluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.
", "doi": "10.7907/4PSM-AS02", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:190", "collection": "thesis", "collection_id": "190", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01152007-080704", "primary_object_url": { "basename": "CompleteThesis.pdf", "content": "final", "filesize": 4146525, "license": "other", "mime_type": "application/pdf", "url": "/190/5/CompleteThesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Chemical Scale Investigations of the Gating Mechanism of Ion Channels", "author": [ { "family_name": "Lee", "given_name": "Lori Wai Hang", "clpid": "Lee-Lori-Wai-Hang" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The studies contained in this dissertation are aimed at utilizing chemistry to understand neurobiology and neuronal communication. Chapters 2 and 3 both address the gating of ion channels, describing structure-function studies to shed light on the gating mechanisms of two classes of ion channels. Chapter 2 studies the gating mechanism of the mechanosensitive channel of small conductance (MscS), which is voltage modulated. Elucidating the mechanism of voltage sensation in MscS may provide insight into how voltage-gated channels translate a change in membrane potential to channel gating. The research discussed in Chapter 2 is aimed at elucidating the role of two arginine residues, in the TM1 and TM2 of MscS, in voltage sensing. We generated two MscS mutants, Arg46Ala and Arg74Ala, to evaluate the effects of \"neutralizing\" the charged side chain on the voltage sensing ability of the channel. The mutants were evaluated using single channel analysis in E. coli spheroplasts. Our preliminary results indicated a potentially significant role for Arg46 in the voltage sensitivity of MscS, however this data set is not extensive due to inconsistency in the spheroplasts preparation.
\r\n\r\nIn Chapter 3, we utilized nonsense suppression to incorporate unnatural amino acids to study the gating of the cation-selective Cys-loop family of ion channel receptors. Specifically, it describes work aimed at elucidating the role of cis-trans isomerization of a proline residue in the gating mechanism of the serotonin-gated 5-hydroxy-tryptamine receptor 3A (5-HT3A) and the nicotinic acetylcholine receptor. A series of proline analgues, of varying cis preference were incorporated at proline 308 in the M2-M3 loop of the 5-HT3A receptor using in vivo nonsense suppression methodology in a Xenopus oocyte expression system. Electrophysiological analysis of the mutant channels revealed a linear relationship between the cis preference of the proline analog and the EC50 of the mutant channel\u2014suggesting that proline 308 may serve as a hinge during the gating 5-HT3A. From these data, we proposed a model of gating for the 5-HT3A receptor. Initial results from similar studies in nAChR suggests that the analogous proline does not play a role in its gating.
\r\n\r\nLastly, Chapter 4 addresses the role of fucose-galactose carbohydrates in learning and memory. It aims to identify lectins to fucose-alpha(1-2)-galactose as well as identify the corresponding glycoproteins bearing fucose-alpha(1-2)-galactose. Chemical probes were synthesized and used to study fucose-alpha(1-2)-galactose binding proteins. One of the probes was used to demonstrate the existence of fucose-alpha(1-2)-galactose binding proteins in hippocampal neurons. Furthermore, initial results from experiments with a photoreactive probe suggested that the design of our probe is sufficient to isolate fucose-alpha(1-2)-galactose binding proteins from the brain. Additionally, we were able to use antibodies specific to fucose-alpha(1-2)-galactose epitopes to examine fucose-alpha(1-2)-galactose bearing glycoproteins in the brain. Overall, results from both studies utilizing chemical probes and molecular probes strongly suggest that the modifications of proteins with fucose-alpha(1-2)-galactose epitopes and the expression of fucose-alpha(1-2)-galactose binding proteins are developmentally regulated.
", "doi": "10.7907/bv54-5p15", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1984", "collection": "thesis", "collection_id": "1984", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05232007-094635", "primary_object_url": { "basename": "bloom_thesis.pdf", "content": "final", "filesize": 1780524, "license": "other", "mime_type": "application/pdf", "url": "/1984/1/bloom_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Hidden Dimensions in Protein Evolution: Stability, Mutational Robustness, and Evolvability", "author": [ { "family_name": "Bloom", "given_name": "Jesse D.", "orcid": "0000-0003-1267-3408", "clpid": "Bloom-Jesse-D" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Adami", "given_name": "Christoph Carl", "orcid": "0000-0002-2915-9504", "clpid": "Adami-C-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Proteins are evolvable in the sense that they are readily able to acquire new or improved functions through the process of mutation and selection. Here, I examine what properties influence the ability of proteins to evolve new functions. I show that proteins with similar biochemical properties can differ substantially in their capacities to withstand mutations and evolve new functions. Specifically, more stable proteins are both more mutationally robust and more evolvable, due to improved tolerance for mutations. This fact can be exploited in protein engineering. I then show how evolutionary theory can be modified to describe how a protein's mutational robustness changes during the normal course of neutral genetic drift. One of the main theoretical predictions is that proteins evolving in larger populations will gain excess stability and mutational robustness, a prediction which I confirm experimentally. Finally, I turn to the question of how neutral genetic drift can alter \"promiscuous\" protein functions that are not under selection. I show that promiscuous functions can change significantly during genetic drift, a phenomenon that may aid in the evolution of beneficial new functions. Overall, this work establishes two mechanisms whereby initially neutral mutations can influence the course of future evolution.", "doi": "10.7907/3MNT-7W93", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:4497", "collection": "thesis", "collection_id": "4497", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-11102006-144154", "primary_object_url": { "basename": "07jheo-Thesis.pdf", "content": "final", "filesize": 6981489, "license": "other", "mime_type": "application/pdf", "url": "/4497/8/07jheo-Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Computational Studies of Orphan G Protein-Coupled Receptors", "author": [ { "family_name": "Heo", "given_name": "Jiyoung", "orcid": "0000-0001-9953-6400", "clpid": "Heo-Jiyoung" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "G protein-coupled receptors (GPCRs) play an essential role in cell communications and sensory functions. Consequently, they are involved in wide variety of diseases and are targets for many drug therapies. Particularly important is the large number of orphan GPCRs, which may play important, albeit unknown, functions in various cells. To understand their respective physiological roles, it is important to identify their endogenous ligands, and to find small molecule ligands that would serve as selective agonists or antagonists. The mas-related gene G protein-coupled receptors (Mrg receptors) belong to the orphan GPCR family, which is expressed in a specific subset of sensory neurons known to detect painful stimuli, suggesting that they could be involved in pain sensation or modulation.
\r\n\r\nThe primary focus of this thesis is to predict the 3D structure and binding site of Mrg receptors and to identify novel ligands that would be potential agonists or antagonists. We predict the 3D structure for the mouse MrgC11 (mMrgC11) and the binding site for five chiral FMRF-NH2 ligands. We correctly predict the relative binding observed for these five ligands. We find that Tyr110 (TM3), Asp161 (TM4), and Asp179 (TM5) are particularly important to binding the ligands. Subsequently, we carry out mutagenesis experiments followed by intracellular calcium release assays that demonstrate the dramatic decrease in activity for the Y110A, D161A, and D179A mutants predicted by our model.
\r\n\r\nThe all-atom molecular dynamics simulation of the mMrgC11/F-(D)M-R-F-NH2 complex structure in explicit water and infinite lipid membrane system shows that some conformational fluctuations are present, but no significant instability is detected, thus validating our structure prediction method.
\r\n\r\nThe virtual screening with the combination of QSPR and docking methods is carried out for the predicted mMrgC11 receptor. The compounds showing the antagonistic effect are identified by competitive functional assays. These hit compounds are certainly good staring points in designing better agonists or antagonists.
\r\n\r\nThe binding site of rat MrgA receptor that shows differential binding between adenine and guanine is also predicted. The predicted binding affinity correlates with the availability of the hydrogen bonds to two Asn residues, which would be primary mutation candidates to validate the structure.
", "doi": "10.7907/rmmr-sj52", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:5046", "collection": "thesis", "collection_id": "5046", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12182006-150535", "primary_object_url": { "basename": "lassila_thesis.pdf", "content": "final", "filesize": 4173076, "license": "other", "mime_type": "application/pdf", "url": "/5046/1/lassila_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Methods for Computational Enzyme Design and Application to the Chorismate-Prephenate Rearrangement", "author": [ { "family_name": "Lassila", "given_name": "Jonathan Kyle", "clpid": "Lassila-Jonathan-Kyle" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The Claisen rearrangement of chorismate to prephenate has become an important model system for developing understanding of enzymatic catalysis as well as for computational treatment of enzyme active sites. This thesis presents general methods for the computational design of enzyme active sites and applies these methods to the design of catalysts for the chorismate-prephenate rearrangement. The computational methods described allow the incorporation of transition-state structures and other small molecules into protein design calculations. These design procedures were tested through redesign of the active site of Escherichia coli chorismate mutase. The six predicted mutations were experimentally characterized and most maintained or increased the catalytic activity of the enzyme. To further investigate the context of the mutations predicted in the calculation and the tolerance of a natural enzyme to secondary active site mutations, extensive substitution experiments were performed. The effect of every amino acid in five active site hydrophobic positions and one N-capping position was evaluated. These experiments clarified some of the strengths and weaknesses of the computational modeling procedure. Finally, attempts to design a completely new enzyme for catalysis of the chorismate-prephenate rearrangement are discussed.", "doi": "10.7907/4xm1-h147", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:5192", "collection": "thesis", "collection_id": "5192", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05212007-164114", "primary_object_url": { "basename": "Alvizo_Thesis_052307.pdf", "content": "final", "filesize": 6107269, "license": "other", "mime_type": "application/pdf", "url": "/5192/1/Alvizo_Thesis_052307.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Computational Protein Design Force Field Optimization: A Negative Design Approach", "author": [ { "family_name": "Alvizo", "given_name": "Oscar", "orcid": "0000-0002-3545-1317", "clpid": "Alvizo-Oscar" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "An accurate force field is essential to computational protein design and protein folding studies. Proper force field tuning is problematic, however, due in part to the incomplete modeling of the unfolded state. The first part of this thesis discusses the optimization of a protein design force field by constraining the amino acid composition of the designed sequences to that of the wild-type protein. According to the random energy model, the unfolded state energies of amino acid sequences with the same composition are identical. Under these constraints, unfolded state energies are inconsequential and any discrepancies between computational predictions and experimental results can be directly attributed to flaws in the force field\u2019s ability to properly account for folded state sequence energies. This aspect of fixed composition design allows for force field optimization by focusing solely on the interactions in the folded state. In addition, the fixed composition requirement imposes a large negative design constraint that is used to ensure fold specificity. Several rounds of fixed composition optimization of the beta-1 domain of protein G yielded force field parameters with significantly greater predictive power: optimized sequences exhibited higher wild-type sequence identity in critical regions of the structure and the wild-type sequence showed an improved Z score. Experimental studies revealed a 24-fold mutant to be stably folded with a melting temperature comparable to that of the wild-type protein.
\r\n\r\nThe second part of the thesis discusses the optimization of HIV protease substrate specificity using a combination of positive and negative design. HIV protease is a homodimeric protein with a symmetrical binding region that recognizes and cleaves asymmetrical substrates that exhibit little sequence homology. The designs attempt to increase specificity towards one of HIV protease\u2019s wild-type targets by optimizing hydrogen bonds and electrostatic interactions using a positive design approach. Explicit negative design is incorporated by modeling predicted mutations on multiple substrates. A scoring function that selects for mutations that pack favorably with the target substrate but result in large steric clashes in alternate substrates is used. A three point mutant was designed and experimentally shown to have increased specificity towards the target substrate.
", "doi": "10.7907/NYVY-7Z76", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:840", "collection": "thesis", "collection_id": "840", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03022007-083916", "primary_object_url": { "basename": "Khidekel_Upload.pdf", "content": "final", "filesize": 7306956, "license": "other", "mime_type": "application/pdf", "url": "/840/1/Khidekel_Upload.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "A Chemoenzymatic Strategy toward Understanding O-GlcNAc Glycosylation in the Brain", "author": [ { "family_name": "Khidekel", "given_name": "Nelly", "clpid": "Khidekel-Nelly" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Posttranslational modification to proteins represents a fundamental mechanism by which protein function is extended and elaborated. In the brain, modifications such as phosphorylation play critical roles in mediating neuronal communication and development. Unique among carbohydrate modifications is the addition of a single monosaccharide, N-acetyl-D-glucosamine, to serine and threonine residues of proteins (O-GlcNAc glycosylation). The modification shares intriguing features with phosphorylation, including its intracellular and dynamic nature. The enzyme responsible for adding the modification to proteins is necessary for life at the single cell level and O-GlcNAc glycosylation has been linked to nutrient sensing, gene expression, and in the brain, to neurodegeneration. Despite tantalizing evidence for the modification\u2019s importance, understanding O-GlcNAc glycosylation has been hampered by insufficient strategies to study it at single-protein level as well as across the proteome. Here we describe the development of a new, chemoenzymatic strategy to facilitate the discovery of O-GlcNAc proteins, as well as the first studies aimed at understanding O-GlcNAc proteome-wide, in the brain.
\r\n\r\nOur approach capitalizes on an engineered enzyme and synthetic unnatural substrate to specifically 'tag' O-GlcNAc-modified proteins for rapid and sensitive detection. We applied the methodology to the discovery of low-abundance, endogenous O-GlcNAc proteins from cells. We also combined the approach with mass spectrometry for the isolation of O-GlcNAc peptides and the mapping of glycosylation sites, the first step toward functional analysis of the modification. Overall, our efforts led to the identification of nearly fifty new O-GlcNAc proteins, several of which serve as targets for mechanistic study. Many of the proteins function in the control of transcription and translation, highlighting the proposed role for O-GlcNAc in regulating gene expression. Additionally, we provide evidence that O-GlcNAc glycosylation is particularly prevalent on proteins at the nerve terminal, or synaptosome, where it may function to control vesicle cycling and neurotransmitter release. Finally, our work has also led to the first bioanalytical, quantitative assays for O-GlcNAc dynamics in both cells and tissue. Here, we have shown that O-GlcNAc is reversible in neuronal tissue and can respond rapidly and robustly to neuronal stimulation in vivo.
", "doi": "10.7907/MT8P-JB95", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1660", "collection": "thesis", "collection_id": "1660", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05072007-135522", "primary_object_url": { "basename": "TOC-Ch1.pdf", "content": "final", "filesize": 2765482, "license": "other", "mime_type": "application/pdf", "url": "/1660/5/TOC-Ch1.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Solving Molecular Recognition Problems with Evolvable Peptide Motifs", "author": [ { "family_name": "Austin", "given_name": "Ryan James", "clpid": "Austin-Ryan-James" } ], "thesis_advisor": [ { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Specific protein-nucleic acid and protein-protein recognition events are frequently mediated through the flexible binding surfaces of these polymers. The functional plasticity and sequence conservation of these surfaces suggests that they are highly evolvable molecular recognition sites. It may therefore be possible to develop discriminate ligands for many protein and nucleic acid targets by directed evolution of consensus ligand scaffolds or motifs. Here we review and present work on the development and use of peptide motifs to evolve high-specificity ligands toward flexible RNA-hairpin and G protein targets. The evolvabilities of these motifs and the compact arrangement of specificity-determining elements in selected sequences, demonstrate the economy of motif-based directed evolution approaches.", "doi": "10.7907/H8BY-M951", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1802", "collection": "thesis", "collection_id": "1802", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05142007-131453", "primary_object_url": { "basename": "Whole_thesis.pdf", "content": "final", "filesize": 8785409, "license": "other", "mime_type": "", "url": "/1802/7/Whole_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Ultrastructural Studies of Two Model Minimal Cells by Electron Cryotomography", "author": [ { "family_name": "Henderson", "given_name": "Gregory Philip", "orcid": "0000-0002-6035-0817", "clpid": "Henderson-Gregory-Philip" } ], "thesis_advisor": [ { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Jensen", "given_name": "Grant J.", "clpid": "Jensen-G-J" }, { "family_name": "Leadbetter", "given_name": "Jared R.", "clpid": "Leadbetter-J-R" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "While most motile bacteria propel themselves with flagella, other mechanisms have been described including retraction of surface-attached pili, secretion of polysaccharides, or movement of motors along surface protein tracks. These have been referred to collectively as forms of \"gliding\" motility. Despite being simultaneously one of the smallest and simplest of all known cells, Mycoplasma pneumoniae builds a surprisingly large and complex cell extension known as the attachment organelle that enables it to glide. Here, three-dimensional images of the attachment organelle were produced with unprecedented clarity and authenticity using state-of-the-art electron cryotomography. The attachment organelle was seen to contain a multi-subunit, jointed, dynamic motor much larger than a flagellar basal body and comparable in complexity. A new model for its function is proposed wherein inchworm-like conformational changes of its electron-dense core are leveraged against a cytoplasmic anchor and transmitted to the surface through layered adhesion proteins.
\r\n\r\nThe hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) were consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes.
", "doi": "10.7907/HK2B-CT58", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2464", "collection": "thesis", "collection_id": "2464", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06052006-155305", "primary_object_url": { "basename": "thesis_new.pdf", "content": "final", "filesize": 5917301, "license": "other", "mime_type": "application/pdf", "url": "/2464/1/thesis_new.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Force Field Development in Protein Design", "author": [ { "family_name": "Zollars", "given_name": "Eric Stafford", "orcid": "0000-0003-0017-9250", "clpid": "Zollars-Eric-Stafford" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Protein design requires the rapid evaluation of very large numbers of equations during the course of a calculation. These equations must represent the important contributors to protein stability in simple and accurate terms. Some physical phenomena are relatively easy to model such as van der Waals forces. Electrostatics and solvation in a protein environment are forces that are more difficult to adequately capture. Additionally, the balance of the terms used must be determined in order to design sequences that fold to stable, specific folds.
\r\n\r\nThe electrostatic interactions within the protein and between the protein and solvent are important in both the stability and function of the protein. The effects of the protein-solvent interactions are evaluated using implicit models that consider the solvent as a bulk. These interactions are quantified using the Poisson-Boltzmann equation that must be solved using discrete numerical methods. We sought to avoid this performance hit by scaling a simpler model of electrostatics, Coulomb's law, to reproduce one aspect of the protein-solvent interaction: solvent screening. By dividing the Coulombic dielectric into two parts and scaling to correlate with the Poisson-Boltzmann results we significantly increased the strength of electrostatics in our force field that led to the design of a more stable engrailed homeodomain.
\r\n\r\nThe second part of this work describes attempts to reparameterize our protein design force field. Many protein mutants have been expressed and biophysically characterized in the literature. We sought to use the measured stabilities of protein mutants in the literature to balance the terms in the force field. While we were able to produce a force field that could reproduce experimental energies, this force field led to unsatisfactory designed sequences. To more fully satisfy the unique conditions of a protein design force field we explored other optimization techniques and found that the balance of the terms in the existing force field is nearly optimal.
", "doi": "10.7907/55br-9a21", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:1978", "collection": "thesis", "collection_id": "1978", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05232006-084250", "primary_object_url": { "basename": "00FrontMatter_v04.pdf", "content": "final", "filesize": 79421, "license": "other", "mime_type": "application/pdf", "url": "/1978/1/00FrontMatter_v04.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Ultrafast Electron Diffraction: Direct Determination of Structural Dynamics of Molecular Excited States", "author": [ { "family_name": "Feenstra", "given_name": "Jonathan Stuart", "clpid": "Feenstra-Jonathan-Stuart" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ultrafast electron diffraction (UED) has been applied to determine the structures of isolated molecules and the dynamics of their excited states. Preceding the experimental accounts is a detailed discussion of the theoretical methodology behind UED in the Caltech labs. The procedure is explained by which electron scattering signal is measured and processed to allow the direct determination of structural dynamics (the signature feature of this experiment). The apparatus itself is also broken down into its component parts and discussed.
\r\n\r\nUED has the capability of studying both ground and excited state systems, which will be demonstrated by example. A number of molecules were studied in their ground states (chlorobenzene, bromobenzene, iodobenzene, 2-fluoropyridine, acetylacetone, benzaldehyde, acetophenone, and methylbenzoate). The structures were determined and compared with structures derived by theoretical calculations and with the results of previous gas electron diffraction inquiries. The molecular structures of 2-fluoropyridine, acetophenone, and methylbenzoate had not been previously experimentally determined. The structure of ground-state acetylacetone is discussed in detail as it represents an old problem involving the influence of intramolecular hydrogen bonding.
\r\n\r\nAcetylacetone, benzaldehyde, and acetophenone were also studied after excitation by a femtosecond laser pulse. Acetylacetone was observed to fragment \u2013 losing the hydroxyl radical. Calculations were performed to further explore the dynamics and mechanism. For excited benzaldehyde and acetophenone, a bifurcation of pathways was structurally resolved. Both molecules have photophysical and photochemical channels from the excited state. The photophysical channels result in the formation of a structure possessing a quinoid ring. The photochemical channels differed \u2013 for benzaldehyde, molecular dissociation resulting in benzene and carbon monoxide, and for acetophenone, homolytic bond cleavage resulting in methyl and benzoyl radicals. The structures of all species were determined as were the time scales involved. Calculations were used to assist in the determination of the excited state decay mechanisms.
", "doi": "10.7907/QCFT-AK63", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2106", "collection": "thesis", "collection_id": "2106", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262006-112358", "primary_object_url": { "basename": "Meyer_Entire_Thesis.pdf", "content": "final", "filesize": 2066524, "license": "other", "mime_type": "application/pdf", "url": "/2106/13/Meyer_Entire_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Exploring Protein Sequence Space Using Computationally Directed Recombination", "author": [ { "family_name": "Meyer", "given_name": "Michelle Margaret", "orcid": "0000-0001-7014-9271", "clpid": "Meyer-Michelle-Margaret" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Evolution has provided us with many protein sequences. However, these sequences represent a very small fraction of the possible sequences. In the laboratory, scientists have explored areas of sequence space not represented by natural proteins both to better understand natural proteins, and to create new proteins with desirable properties. The principle mechanism used to explore protein sequence space is mutagenesis. However, recombination of homologous genes can also explore regions of sequence space rich with folded and functional proteins.
\r\n\r\nIn this work we demonstrated using a beta-lactamase model system that a computation energy function (SCHEMA) can predict which of the chimeras made by recombining distantly related proteins are likely to fold. SCHEMA uses protein sequence and structure information to identify pairwise amino acid interactions disrupted by recombination. Using SCHEMA we designed libraries of chimeric beta-lactamases. These libraries were intended to have a high fraction of folded variants, while incorporating many amino acid substitutions compared with the parental proteins. The chimeras in these libraries were characterized to determine whether they retain the parental function and what new substrate specificities could be obtained.
\r\n\r\nTo identify critical variables for determining whether a chimera functions, we used logistic regression analysis to analyze functional and nonfunctional chimeras. From this analysis it is apparent that both two-body (pairwise) and one-body terms play a significant role in determining whether a chimera functions. We also used random mutagenesis to restore functionality to nonfunctional chimeras showing that a thermostabilizing mutation can rescue approximately 5% of the nonfunctional chimeras. The one-body terms that appear significant for determining whether a chimera functions are not explicitly counted by SCHEMA when predicting chimera folding. To estimate the effects on chimera folding represented by the one-body terms, we developed an additional measure to predict chimera folding based on just the chimera amino acid sequence and a multiple sequence alignment of homologous proteins. This measure is predictive of chimera folding alone, and when combined with the pairwise SCHEMA energy increases the accuracy of the folding predictions compared to SCHEMA.
", "doi": "10.7907/YJPE-CD11", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3672", "collection": "thesis", "collection_id": "3672", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09202008-110622", "primary_object_url": { "basename": "Doss_rm_2006.pdf", "content": "final", "filesize": 19448639, "license": "other", "mime_type": "application/pdf", "url": "/3672/1/Doss_rm_2006.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Programmable Oligomers for DNA Recognition", "author": [ { "family_name": "Doss", "given_name": "Raymond Michael", "clpid": "Doss-Raymond-Michael" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "As the amount of information about the genetic construct of the human body continues to grow, the ability to manipulate genes via the use of synthetic molecules becomes an increasingly attractive concept. Polyamides developed in the Dervan Lab are capable of doing just this by binding in the minor groove of DNA in a highly specific manner. Not only are polyamides able to specifically target sequences of DNA, but they are able to do so at affinities which make them competitive with endogenous transcriptional machinery.
\r\n\r\nThe complex nature of the DNA minor groove structure, however, has forced the evolution of traditional imidazole, pyrrole and hydroxypyrrole polyamides into newly developed oligomers \u2014 compounds which have been shown to bind sequences of DNA that have been traditionally difficult to target. In going from polyamides to oligomers, these compounds have seen a variety of changes brought about by the search for ring systems capable of conveying improved binding properties. Several new recognition elements have been uncovered and characterized with respect to their DNA affinity and specificity. Experiments testing the capabilities of these oligomers have shown that such compounds demonstrate great potential for targeting many new, biologically relevant sequences of DNA thus showing promise as potential 2nd generation therapeutics.
\r\n", "doi": "10.7907/F0X3-Q612", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3671", "collection": "thesis", "collection_id": "3671", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09202008-110124", "primary_object_url": { "basename": "McGarvey_t_2006.pdf", "content": "final", "filesize": 8270442, "license": "other", "mime_type": "application/pdf", "url": "/3671/1/McGarvey_t_2006.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Ultra-Sensitive Absorption Measurements through Cavity-Enhanced Spectroscopy", "author": [ { "family_name": "McGarvey", "given_name": "Raymond Timothy James", "clpid": "McGarvey-Raymond-Timothy-James" } ], "thesis_advisor": [ { "family_name": "Mabuchi", "given_name": "Hideo", "clpid": "Mabuchi-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Yang", "given_name": "Changhuei", "clpid": "Yang-Changhuei" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Mabuchi", "given_name": "Hideo", "clpid": "Mabuchi-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The desire to increase the sensitivity of solution-based absorption spectroscopy is motivated by the need for label-free biosensing (which provides a more authentic indication of the state of a biological system) and by the usefulness of characterizing the kinetics of biologically-relevant reactions (which may not be accurately characterizable at reagent concentrations required by standard methods. There are a number of techniques by which such increasingly sensitive measurements have been made, including cavity ringdown spectroscopy, incoherent cavity-enhanced spectroscopy, microsphere-based whispering-gallery mode sensing,and our cavity-enhanced measurements, which are the most sensitive to date and which can be conducted in real time with high bandwidth. Our current device has a demonstrated detection threshold of 1.7x 10^{-7}/sqrt{Hz} (4.36x10^{-6}cm^{-1}), which could with further technical work be improved to a shot-noise limited sensitivity of 1.93x 10^{-10}/sqrt{Hz} (1.06x10^{-8}cm^{-1}). The latter would correspond to an average of 700 strong absorbers (epsilon = 10^5 M^{-1}cm^{-1}) in the optical beam volume. The shot-noise limited detection threshold of our measurement method could potentially be improved by up to two orders of magnitude by incorporating state-of-the-art optical mirrors. With such mirrors, cavity-enhanced absorption experiments performed with gas-phase samples have previously demonstrated single molecule sensitivity. We have established that solution-based cavity-enhanced absorption measurements are more sensitive than standard single-pass measurements by the predicted enhancement factor for our present device (~ 20,000). These measurements provide the proof-of-principle for solution-based, cavity-enhanced spectroscopy and serve as the intermediate step towards the attainment of the theoretical sensitivity of this technique. We believe that this device will be of broad interest to the scientific community, because it is presently the most sensitive solution-based spectroscopic device. It can make real-time absorption measurements which would allow monitoring of the kinetics of chemical reactions in which the spectral properties of reactants change by even a small amount, and, near its theoretical limit of sensitivity (given currently available mirrors), such a device could potentially resolve single-molecule absorption events on the sub-millisecond timescale and below.\r\n", "doi": "10.7907/CGYD-6J27", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:839", "collection": "thesis", "collection_id": "839", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03022006-172355", "primary_object_url": { "basename": "Cashin-Amanda-thesis-2006.pdf", "content": "final", "filesize": 13486878, "license": "other", "mime_type": "application/pdf", "url": "/839/49/Cashin-Amanda-thesis-2006.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Chemical Scale Investigations of Drug-Receptor Interactions at the Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Cashin", "given_name": "Amanda Leigh", "clpid": "Cashin-Amanda-Leigh" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biological signaling pathways employ a vast array of integral membrane proteins that process and interpret the chemical, electrical, and mechanical signals that are delivered to cells. Among these proteins, ligand gated ion channels (LGIC) are therapeutic targets for Alzheimer\u2019s disease, Schizophrenia, drug addiction, and learning and memory. High-resolution structural data on neuroreceptors are only just becoming available, yet the functional importance of particular structural features can be challenging.
\r\n\r\nThe primary focus of the present work is to gain a chemical scale understanding of the ligand-receptor binding determinants of LGICs. In particular, these studies explore drug-receptor interactions at the nicotinic acetylcholine receptor (nAChR), the most extensively studied members of the Cys-loop family of LGICs. The present study utilizes in vivo nonsense suppression methodology to perform chemical scale investigations of nAChR agonist activity.
\r\n\r\nThe binding of three distinct agonists\u2013-acetylcholine (ACh), nicotine, and epibatidine--to the nicotinic acetylcholine receptor (nAChR) has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp a149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond.
\r\n\r\nNonsense suppression was also utilized to probe the importance of residues outside of the binding box in nAChR function. These studies demonstrate a structural role of the highly conserved aD89 residue in stabilizing the agonist binding site near aW149. In addition to outer shell residue, aK145 is shown to be important for proper nAChR function. In combination with additional evidence from other recent advances, this site is proposed to be important in initiating the nAChR channel gating pathway.
\r\n\r\nResidues outside the aromatic binding site were also examined through computational protein design studies. Results from these studies identify outer shell mutations 116Q and 57R (AChBP numbering) that enhance nAChR specificity for nicotine, over ACh and epibatidine compared to wild-type receptors.
\r\n\r\nFinally, a series of cationic polyamides were shown to enhance polyamide affinity while maintaining specificity by varying the number, relative spacing, and linker length of aminoalkyl side chains.
", "doi": "10.7907/RX9T-1069", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:595", "collection": "thesis", "collection_id": "595", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02102006-100744", "primary_object_url": { "basename": "JessicaMaoThesis.pdf", "content": "final", "filesize": 9439742, "license": "other", "mime_type": "application/pdf", "url": "/595/1/JessicaMaoThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Applications of Computational Protein Design", "author": [ { "family_name": "Mao", "given_name": "Jessica", "clpid": "Mao-Jessica" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Computational protein design determines the amino acid sequence(s) that will adopt a desired fold. It allows the sampling of a large sequence space in a short amount of time compared to experimental methods. Computational protein design tests our understanding of the physical basis of a protein\u2019s structure and function, and over the past decade, has proven to be an effective tool.
\r\n\r\nWe report the diverse applications of computational protein design with ORBIT (Optimization of Rotamers by Iterative Techniques). We successfully utilized ORBIT to construct a reagentless biosensor for nonpolar ligands on the maize non-specific lipid transfer protein, by first removing native disulfide bridges. We identified an important residue position capable of modulating the agonist specificity of the mouse muscle nicotinic acetylcholine receptor (nAChR) for its agonists: acetylcholine, nicotine, and epibatidine. Our efforts on enzyme design produced a lysozyme mutant with ester hydrolysis activity, while progress was made toward the design of a novel aldolase.
\r\n\r\nComputational protein design has proven to be a powerful tool for the development of novel and improved proteins. As we gain a better understanding of proteins and their functions, protein design will find many more exciting applications.
\r\n", "doi": "10.7907/13A4-Z652", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3265", "collection": "thesis", "collection_id": "3265", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08292005-210930", "primary_object_url": { "basename": "TW_Thesis.pdf", "content": "final", "filesize": 2367366, "license": "other", "mime_type": "application/pdf", "url": "/3265/1/TW_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "A Chemical-Scale Study on the Ligand-Binding Site of a Serotonin-Gated Ion Channel", "author": [ { "family_name": "Mu", "given_name": "Tingwei", "orcid": "0000-0002-6419-9296", "clpid": "Mu-Tingwei" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Signal transmission is a combination of electrical and chemical processes. Upon binding neurotransmitters, ligand-gated ion channels open to allow ion flux, which converts chemical signals to electrical signals. In this thesis, experiments in conjunction with computations are utilized to study the mechanism of the ligand-binding process.
\r\n \r\nThe target receptor is a serotonin-gated chloride channel, the MOD-1 receptor. From the viewpoint of a chemist, we explore the specific orientation of the agonist inside the binding pocket and the specific non-covalent interactions responsible for binding. In Chapter 3, computational chemistry is used to build a homology model of MOD-1 using the acetylcholine binding protein template. We proceed to dock the agonist into the binding pocket. The binding pattern from the model provides guidance for the ensuing experimental studies.
\r\n\r\nUnnatural amino acid mutagenesis is a powerful tool to modify the structure of the protein at the chemical level. Systematic perturbations can be introduced at a specific amino acid. Therefore, specific non-covalent interactions, such as hydrogen bonding and cation-pi interactions can be probed. In Chapter 2, we prove that cation-pi interactions between the agonist serotonin and Trp 226 in loop C of MOD-1 play a key role in binding the ligand. Surprisingly, this cation-pi site in MOD-1 is different from that in the serotonin type 3 receptor although these two receptors both bind serotonin, and they are highly homologous. In Chapter 4, we further show that hydrogen bonds between serotonin and Gln 228 and Asn 223 in MOD-1 are important in the binding process. Both conventional and unnatural amino acid mutagenesis are used in conjunction with serotonin analogues. The results from these thorough structure-function studies confirm aspects of the hydrogen bond pattern described in the model.
\r\n\r\nIn Chapter 5, we apply another strategy called the tethered agonist approach to further probe the agonist binding site. This is another elegant example of the effectiveness of the nonsense suppression method.
", "doi": "10.7907/VSEF-4856", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:941", "collection": "thesis", "collection_id": "941", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03132006-112152", "primary_object_url": { "basename": "Complete_thesis.pdf", "content": "final", "filesize": 9705860, "license": "other", "mime_type": "application/pdf", "url": "/941/5/Complete_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigations of Ion Channels with Computational Simulations and Biochemical Experiments", "author": [ { "family_name": "Spronk", "given_name": "Steven Adrian", "clpid": "Spronk-Steven-Adrian" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chapter one describes studies of the voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance, MscS. A detailed picture of water and ion properties in small pores is important for understanding the behavior of biological ion channels. Several recent modeling studies have shown that small, hydrophobic pores exclude water and ions even if they are physically large enough to accommodate them, a mechanism called hydrophobic gating. This mechanism has been implicated in the gating of several channels, including MscS. Although the pore in the crystal structure of MscS is wide and was initially hypothesized to be open, it is lined by hydrophobic residues and may represent a nonconducting state. Molecular dynamics simulations were performed on MscS to determine whether or not the structure can conduct ions. Unlike previous simulations of hydrophobic nanopores, electric fields were applied to this system to model the transmembrane potential, which proved to be important. Although simulations without a potential resulted in a dehydrated, occluded pore, the application of a potential increased the hydration of the pore and resulted in current flow through the channel. The calculated channel conductance was in good agreement with experiment. Therefore, it is likely that the MscS crystal structure is closer to a conducting than to a nonconducting state.
\r\n\r\nChapter two describes work toward a method using protein transduction domains (PTDs) to deliver tRNA to cultured mammalian cells. In vivo incorporation of unnatural amino acids using nonsense suppression is a powerful technique to study proteins. However, one challenge to the method is that the amount of unnatural protein that can be produced is directly limited by the amount of unnatural aminoacyl-tRNA presented to the cellular translation machinery. Therefore, the success of this technique depends heavily on the ability to deliver aminoacyl-tRNA, which is produced in vitro, into cells. Currently, the most commonly used system involves injection of a Xenopus oocyte. It is desirable to transfer the technology to a mammalian expression system, but because mammalian cells are so much smaller than oocytes, injection is not a practical delivery method, so other techniques must be utilized. An intriguing possibility is the use of PTDs, small peptides that greatly enhance the internalization of extracellular material. Several PTD-based approaches for tRNA delivery were attempted: covalent ligation of tRNA to a PTD, noncovalent complexation of tRNA and PTDs, and production of a fusion protein containing a PTD and a tRNA-binding domain. However, none of these methods was useful in delivering tRNA into mammalian cells in culture.
\r\n\r\nChapter three describes efforts to develop a high throughput assay for gating of the mechanosensitive channel of large conductance, MscL. The bacterial ion channel MscL is an ideal starting point for understanding the molecular basis of mechanosensation. However, current methods for the characterization of its mutants, patch clamp and bacterial growth analysis, are difficult and time consuming, so a higher throughput method for screening mutants is desired. We have attempted to develop a fluorescence assay for detecting MscL activity in synthetic vesicles. The assay involved the separation of two solutions\u2014one inside and one outside the vesicles\u2014that are separately nonfluorescent but fluorescent when mixed. It was hoped that MscL activity due to downshock of the vesicles would bring about mixing of the solutions, producing fluorescence. The development of the assay required the optimization of several variables: the method for producing a uniform vesicle population containing MscL, the fluorescence system, and the lipid and protein composition of the vesicles. However, no MscL activity was ever detected even after optimization, so the assay was not fully developed. The probable cause of the failure was the inability of current techniques to produce a sufficiently uniform vesicle population.
", "doi": "10.7907/Z399-V061", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2528", "collection": "thesis", "collection_id": "2528", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06092006-062410", "primary_object_url": { "basename": "thesis3.pdf", "content": "final", "filesize": 7605036, "license": "other", "mime_type": "application/pdf", "url": "/2528/1/thesis3.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Applications of Bulky Rhodium(III) Intercalators for the Recognition of DNA Mismatches", "author": [ { "family_name": "Hart", "given_name": "Jonathan Ross", "orcid": "0000-0002-3905-225X", "clpid": "Hart-Jonathan-Ross" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "orcid": "0000-0002-6610-4414", "clpid": "Peters-J-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The recognition of DNA base mismatches is of considerable interest for both the diagnosis and treatment of mismatch repair-deficient cancers. Two new mismatch recognition complexes have been synthesized. The first, [Rh(bpy)2(phzi)]3+ (phzi=benzo[a]phenazine-5,6-quinone diimine), recognizes DNA mismatches with high specificity and affinity, 1 x 107 Mm-1, two orders of magnitude stronger than [Rh(bpy)2(chrysi)]3+ (chrysi=chrysene-5,6-quinone diimine), the parent complex that binds single thermodynamically-destabilized base-mismatch sites in duplex DNA. The second, [Rh(bqdi)2(chrysi)]3+, is able to recognize more stable mismatches such as the G-G mismatch.
\r\n\r\nThese complexes have been applied in a variety of ways. A method has been developed for the discovery of new single nucleotide polymorphisms, SNPs, within a sequence of interest amplified from pooled genomic DNA. SNPs are readily detected using these mismatch selective molecules without false positives; allele frequencies as low as 0.05 can be detected.
\r\n\r\nUpon photoexcitation, the rhodium(III) diimine complexes cleave DNA by hydrogen atom abstraction from the sugar to yield 3'-phosphate terminated DNA that is inactive for enzymatic modification. This 3'-phosphate can be removed using T4-polynucleotide kinase opening up the possibility of enzymatic modification at the site of rhodium cleavage. The cleavage site can be fluorescently labeled. Terminal transferase can also be used to attach a homopolymer tail tagging the damage site, allowing the amplification of the DNA up to the damaged site.
\r\n\r\nThis assay can also be employed towards the development of early cancer diagnostics. Some cancers are deficient in the repair of DNA base mismatches. As a consequence, these cells have an increased number of mismatches within their genome. These mismatches in extracted genomic DNA were cleaved using mismatch-specific rhodium complexes. The cleavage sites were labeled with radioactivity, allowing the number of mismatch sites to be quantitated. A significant number of sites were cleaved in the mismatch repair deficient DU145 cell line, 1 base/3000 bp, while no sites were cleaved in the mismatch repair proficient cell line SW620. This method may present a new method for the detection of mismatch repair deficiency.
\r\n\r\nThese mismatch-specific complexes also are shown to have an antiproliferative effect on mismatch repair deficient cell lines. Mismatch repair deficiency is a contributing factor in both hereditary and sporadic human cancers. Both [Rh(bpy)2(chrysi)]Cl3 and [Rh(bpy)2(phzi)]Cl3 show a stronger antiproliferative effect against MMR deficient cells than proficient cells. Effects of stereoisomers, incubation time, and UV irradiation are also demonstrated.
", "doi": "10.7907/sh3b-2f25", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:10", "collection": "thesis", "collection_id": "10", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01032005-161114", "primary_object_url": { "basename": "Complete_Thesis.pdf", "content": "final", "filesize": 3591529, "license": "other", "mime_type": "application/pdf", "url": "/10/8/Complete_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Peptide Modulators of G Protein Signaling", "author": [ { "family_name": "Ja", "given_name": "William Wei-Hua", "orcid": "0000-0002-4003-7356", "clpid": "Ja-William-Wei-Hua" } ], "thesis_advisor": [ { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The hundreds of transmembrane proteins that make up the superfamily of G protein-coupled receptors (GPCRs) mediate signaling from an enormous variety of extracellular stimuli\u2014including odorants, pheromones, peptides, lipids, and neurotransmitters\u2014to intracellular heterotrimeric G proteins. The identification of specific modulators of G protein signaling is highly relevant to drug discovery; approximately 50% of currently marketed drugs target a GPCR. Here, we use mRNA display to identify novel and potent peptide ligands for G protein targets. mRNA display is a robust technique that facilitates the isolation of peptides with specific activities (e.g., binding to a target of interest) from large libraries containing trillions of unique molecules. We first targeted the heterotrimeric G protein, Gi(alpha)1, with peptide combinatorial libraries. Isolated peptides bind with high affinity to Gi(alpha)1 and can potentially affect downstream signaling in a pathway-specific manner. A potent peptide core motif interacting with G(alpha) subunits was identified and used to construct new mRNA display libraries for the isolation of class- and/or state-specific G(alpha)-binding peptides. We have also identified a novel peptide (the RWR motif) that interacts with the Drosophila GPCR, Methuselah. These peptides are potent antagonists to Methuselah-mediated signaling and, as mutants of Methuselah are associated with longevity, may be useful in lifespan and aging studies of the fruit fly. Overall, these efforts demonstrate the successful use of mRNA display as an efficient and facile method for generating new solutions to molecular design problems.", "doi": "10.7907/WMQ9-8086", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2422", "collection": "thesis", "collection_id": "2422", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06032005-113204", "primary_object_url": { "basename": "Meinhold_Thesis.pdf", "content": "final", "filesize": 4426788, "license": "other", "mime_type": "application/pdf", "url": "/2422/2/Meinhold_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Cytochrome P450 BM-3 for Selective Hydroxylation of Alkanes", "author": [ { "family_name": "Meinhold", "given_name": "Peter", "clpid": "Meinhold-Peter" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Labinger", "given_name": "Jay A.", "orcid": "0000-0002-1942-9232", "clpid": "Labinger-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cytochromes P450 are of potential synthetic value because they hydroxylate a large array of substrates, often with high regio- and enantioselectivity. In contrast to most P450s, the BM-3 variant from Bacillus megaterium is soluble, easily expressed in E. coli, and does not require additional electron transfer proteins. A highly efficient enzyme for its preferred reaction, hydroxylation of medium-chain (C12 to C18) fatty acids, BM-3 is a good candidate for engineering for applications requiring activity on other substrates.
\r\n\r\nUsing iterations of random mutagenesis, recombination, and high throughput screening, we engineered P450 BM-3 mutants to hydroxylate linear alkanes as short as propane. Activity towards linear alkanes was further increased by changing two key active site residues. The resulting mutants hydroxylate linear alkanes with high regioselectivity and, notably, enantioselectivity.
\r\n\r\nWe further engineered these enzymes with guidance from the crystal structure of substrate-bound P450 BM-3. Eleven active-site residues were chosen for saturation mutagenesis, and the resulting mutants were screened for improved activity towards alkanes, as measured by total product formation. Substitutions at these positions generally did not affect correct folding of the enzyme, and a large fraction of folded proteins retained similar levels of activity as their predecessor. Moreover, several of the 11 selected amino acid substitutions yielded mutants that were both more active and produced various combinations of product regioisomers.
\r\n\r\nRecombination of these beneficial active-site mutations generated BM-3 variants that catalyze: (a) regio- and enantioselective hydroxylation of linear alkanes; (b) terminal hydroxylation of linear alkanes; (c) regio- and enantioselective hydroxylation of heterocyclic compounds; and (d) ethane hydroxylation.
\r\n\r\nThe selective conversion of ethane to ethanol, not previously reported for any P450, is catalyzed by the most active mutant from this library. In nature, this reaction is solely observed for methane monooxygenases (MMOs) and related enzymes in alkane-assimilating bacteria.
\r\n\r\nAdditionally, we have found that the reductase domain can be engineered to increase the efficiency of these reactions. Our progress in converting BM-3 from a fatty-acid hydroxylase into an enzyme able to selectively hydroxylate smaller alkanes, including ethane, is an important step towards our ultimate goal, achieving selective BM-3 catalyzed conversion of methane to methanol.
", "doi": "10.7907/aats-ca30", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1266", "collection": "thesis", "collection_id": "1266", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04042005-142719", "primary_object_url": { "basename": "Premal_Shah_Thesis_Final.pdf", "content": "final", "filesize": 1928056, "license": "other", "mime_type": "application/pdf", "url": "/1266/1/Premal_Shah_Thesis_Final.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Advances in Force Field Development and Sequence Optimization Methods for Computational Protein Design", "author": [ { "family_name": "Shah", "given_name": "Premal S.", "clpid": "Shah-Premal-S" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The overall goals of computational protein design range from designing new protein folds and protein-protein interfaces to the de novo design of enzymes. All goals require that two equally challenging components of computational protein design be addressed. First, the physical model that describes a protein\u2019s intermolecular and intramolecular interactions must be accurate. Second, energetically optimal amino acid sequences must be identified from an enormous number of possibilities. This thesis describes work that makes progress in both these arenas. In addition, the effectiveness and applicability of computational protein design is demonstrated by tackling challenging design problems.
\r\n\r\nImprovements to the physical model have been made by developing a more accurate method for calculating rotamer (amino acid side-chain conformation) surface areas for use in our surface area-based hydrophobic solvation term. With this method, surface area errors were decreased dramatically and the experimental stabilities of proteins generated from computationally predicted sequences were improved. Also, our direct surface area calculation approach significantly reduced the compute time required for sequence optimization using dead-end elimination (DEE)-based algorithms.
\r\n\r\nAlthough DEE-based algorithms have been effectively used for many challenging design problems, the daunting task of sequence optimization can cause even the most efficient DEE-based methods to fail. We developed a sequence optimization technique called Vegas that combines elements of non-DEE-based as well as DEE-based algorithms. For design problems that were already tractable using DEE-based methods, Vegas delivered the GMEC in significantly less time. In cases where DEE-based algorithms stalled and failed to deliver the GMEC, Vegas produced an answer that, at the time, was better than any other algorithm. This is illustrated by Vegas\u2019 solution to a challenging problem: the full sequence design of a 51-residue fragment of the Drosophila engrailed homeodomain (ENH). We generated a variant of ENH predicted by Vegas and compared its thermodynamic properties with a protein obtained using a Monte Carlo search. We found that the thermodynamic properties of the two molecules were identical. We also solved the solution structure of the Vegas-based molecule using nuclear magnetic resonance (NMR) spectroscopy and found that it folded accurately into the target fold.
\r\n\r\nObtaining water soluble variants of membrane proteins might alleviate some of the problems encountered when working with them and facilitate our understanding of the different forces contributing to protein stabilities in membranes. We made progress in developing an automated design scheme that can generate water soluble variants of membrane proteins. We analyzed and compared the surfaces of membrane proteins and water soluble proteins, and developed a metric for altering membrane protein surfaces. Using this metric, we can design membrane protein surfaces using the ORBIT suite of protein design algorithms and convert them to those resembling water soluble protein surfaces. We tested this strategy on two proteins and although we have not been completely successful, we have established rules and guidelines that will aid future efforts towards achieving this goal.
", "doi": "10.7907/mg9x-s593", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2148", "collection": "thesis", "collection_id": "2148", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272005-121337", "primary_object_url": { "basename": "sarisky_phd.pdf", "content": "final", "filesize": 1471982, "license": "other", "mime_type": "application/pdf", "url": "/2148/1/sarisky_phd.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Exploration of the Determinants of Protein Structure and Stability by Protein Design", "author": [ { "family_name": "Sarisky", "given_name": "Catherine Ann", "orcid": "0000-0002-7692-2517", "clpid": "Sarisky-Catherine-Ann" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Optimization of Rotamers by Iterative Techniques (ORBIT) has been used to calculate novel sequences for several small proteins. A partial sequence design (20 of 28 residues) is described for the zinc finger Zif268 (beta-beta-alpha) motif. The designed peptide folds without a metal cofactor, despite its small size and the avoidance of the disulfides and unnatural amino acids that are often used to stabilize peptide structures. The utility of ORBIT for predicting the relative stabilities of a series of beta-beta-alpha peptides was investigated. A good correlation between theoretical and experimental stabilities was observed except when the turn residues were changed. This observation led to the discovery that some of these peptides had an unexpected turn conformation. This information was used to design a peptide that is more stable than the original peptide sequence produced with ORBIT.
\r\n\r\nThe tolerance of ORBIT for altered backbone coordinates was investigated using the protein domain G-beta1. It was determined that altering the coordinates of the backbone template used in ORBIT altered the sequences selected, but that the fold did not change as a result. The G-beta1 domain was also used to parameterize a methionine inclusion penalty, allowing the inclusion of methionine in ORBIT design calculations while preventing indiscriminate inclusion of methionine at sites where a less flexible side-chain will fit.
\r\n\r\nLastly, some preliminary work on using ORBIT to design DNA binding interfaces is discussed.
", "doi": "10.7907/RBSA-R089", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2099", "collection": "thesis", "collection_id": "2099", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262005-163739", "primary_object_url": { "basename": "EntireThesis.pdf", "content": "final", "filesize": 15492218, "license": "other", "mime_type": "", "url": "/2099/9/EntireThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "In Vitro Selection of RNA Binding Peptides", "author": [ { "family_name": "Takahashi", "given_name": "Terry Torao", "clpid": "Takahashi-Terry-Torao" } ], "thesis_advisor": [ { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "RNA is recognized to play an increasing number of roles in the cell: transcription regulation, translation, and catalysis. Peptides that bind RNA would therefore be useful as biochemical tools and lead compounds for therapeutics. Existing genetic methods of isolating RNA binding peptides are prone to biases and can only search millions of sequences. In vitro selections using mRNA display provide an avenue to discover specific, high affinity peptides that bind to any RNA target from libraries composed of trillions of molecules.
\r\n\r\nHere, we describe initial experiments to optimize the mRNA display selection cycle for the isolation of RNA binding peptides. We use this optimized cycle to show that enrichment of specific sequences is possible using mRNA display, and select mutants of the lambda N peptide which bind in a different conformation than wild-type. Characterization of these peptides demonstrates that affinity is not enough for in vivo activity; binding in a correct conformation is also important.
\r\n\r\nBased on these experiments, we designed a strategy to isolate RNA binding peptides to targets for which no natural ligand is known. We test this strategy and isolate peptides that bind to functionally important domains of telomerase RNA with nanomolar affinity and high specificity. Using mutagenic PCR and additional rounds of selection, we increase the specificity of several peptides for telomerase RNA and also isolate other peptides which bind an important domain of the Hepatitis C Virus internal ribosome entry site.
", "doi": "10.7907/9X0X-PG47", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:3833", "collection": "thesis", "collection_id": "3833", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09302004-090155", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 2083079, "license": "other", "mime_type": "application/pdf", "url": "/3833/2/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structural and Functional Studies of Jamm Domain Proteins and Their Role in the Ubiquitin System", "author": [ { "family_name": "Ambroggio", "given_name": "Xavier Ignacio", "clpid": "Ambroggio-Xavier-Ignacio" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif is a conserved amino acid sequence, EX(n)HS/THX(7)SXXD, found in proteins from all domains of life. Eukaryotic proteins possesing a JAMM motif are responsible for the selective hydrolysis of iso-peptide linkages involving ubiquitin and ubiquitin-like proteins and often exist as subunits of large complexes. The iso-peptidase activity of JAMM proteins plays a major role in key points of regulation in the ubiquitin system. In particular, the JAMM motif of CSN5 of the COP9 signalosome is responsible for the cleavage of the ubiquitin-like Nedd8 from SCF ubiquitin ligases. A homolog of CSN5 in the lid subcomplex of the 19S proteasome regulatory particle, Rpn11, cleaves ubiquitin from proteasome substrates as they are processed by the proteasome. In order to understand the mechanism underlying iso-peptide bond hydrolysis by the JAMM motif, we have solved the crystal structure of a JAMM domain protein from Archaeoglobus fulgidus, AfJAMM. The JAMM motif forms a thermolysin-like active site on a cytidine deaminase fold. We have demonstrated through biochemical analysis of mutations in the JAMM motif of Csn5 that the mechanism of hydrolysis is similar to that of thermolysin. To achieve an integrated understanding of a JAMM domain protein within its cognate complex, we have purified and crystallized the lid subcomplex of the 19S proteasome regulatory particle for structural studies.", "doi": "10.7907/EVW4-CC51", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:4962", "collection": "thesis", "collection_id": "4962", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12122004-215151", "primary_object_url": { "basename": "Shapovalov_00_title.pdf", "content": "final", "filesize": 29183, "license": "other", "mime_type": "application/pdf", "url": "/4962/2/Shapovalov_00_title.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Mechanosensitive Channels of Bacteria: Structure and Function. Electrophysiology as a High Resolution Technique of Ion Channel Study", "author": [ { "family_name": "Shapovalov", "given_name": "George G.", "orcid": "0000-0001-9702-9317", "clpid": "Shapovalov-George-G" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mabuchi", "given_name": "Hideo", "orcid": "0000-0002-5156-7678", "clpid": "Mabuchi-H" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "Mechanosensitive (MS) ion channels commonly play a role of transducers converting mechanical stimuli into electrical or chemical signaling, thus allowing the cell to regulate its behavior in response to changing environment conditions. MS channels participate in sensation of sound and orientation in inner ear (hair cells), in touch sensation and in osmoregulation of bacteria. Structure of bacterial MS channels of large (MscL) and small (MscS) conductance has been recently solved at atomic resolution, stimulating various structural and functional studies. In this work author presents series of experiments enhancing an understanding of mechanosensation in bacteria. In Chapter 2 author performs cysteine cross-linking experiments suggesting asymmetric gating pattern of Tb-MscL ion channel. Chapters 3 and 4 establish a possibility of successful synthesis of fully functional Tb- and Ec-MscL proteins displaying a phenotype identical to recombinant channels. Studies in Chapter 5 and Appendix 1 extend the resolution of single-channel patch clamping technique, and describe a fine structure of MS channel gating by collecting and characterizing intersubstate transitions.", "doi": "10.7907/XZ69-9A14", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:9", "collection": "thesis", "collection_id": "9", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01032005-142802", "primary_object_url": { "basename": "Prologue.pdf", "content": "final", "filesize": 77056, "license": "other", "mime_type": "application/pdf", "url": "/9/7/Prologue.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structural and Biochemical Characterization of the Vitamin B\u2081\u2082 ABC Transporter, BtuCD-F", "author": [ { "family_name": "Borths", "given_name": "Elizabeth Loraine", "clpid": "Borths-Elizabeth-Loraine" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "BtuCD-F is a binding protein-dependent ABC transporter system that uses the power of ATP hydrolysis to pump vitamin B\u2081\u2082 into the cytoplasm of E. coli. The crystal structure of BtuF, the protein that binds vitamin B\u2081\u2082 and delivers it to the transporter, BtuCD, has been solved by x-ray crystallography. BtuF is a bi-lobed protein and B\u2081\u2082 is bound in a deep cleft formed at the interface between the two lobes. A stable complex between BtuF and BtuCD is demonstrated to form in vitro and was modeled using the individual crystal structures. Two conserved surface glutamates from BtuF may interact with conserved arginine residues on the periplasmic surface of the BtuCD transporter, playing a role in docking and the transmission of conformational changes.
\r\n\r\nBtuCD has also been reconstituted in vitro into proteoliposomes. In the presence of ATP, BtuCD proteoliposomes can mediate uptake of vitamin B\u2081\u2082 in a BtuF dependent fashion. In the absence of ATP, B\u2081\u2082 appears to become sequestered between BtuF and BtuCD. The ATPase activity of BtuCD was examined in proteoliposomes as well as in detergent solution. BtuCD has a significant basal rate of hydrolysis under all conditions tested, and B\u2081\u2082-bound and apo-BtuF can stimulate that rate. Interestingly, the rate of ATP hydrolysis, as well as the effect of BtuF, vitamin B\u2081\u2082 and sodium ortho-vanadate on that rate, is different in each detergent and lipid environment. These results indicate that ABC transporters are highly sensitive to their environment and underline the importance of detergent or lipid choice in functional reconstitution and membrane protein crystallization experiments. Our results lead us to propose a revised model of the ABC transport cycle.
", "doi": "10.7907/kkkr-tk81", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2456", "collection": "thesis", "collection_id": "2456", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06052005-164552", "primary_object_url": { "basename": "01Titlepage.pdf", "content": "final", "filesize": 62941, "license": "other", "mime_type": "application/pdf", "url": "/2456/1/01Titlepage.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Design of Sequence-Specific DNA Intercalators", "author": [ { "family_name": "Fechter", "given_name": "Eric James", "clpid": "Fechter-Eric-James" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind specific DNA sequences may have powerful therapeutic applications by influencing the mechanisms of abnormal gene expression. Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) specifically bind the minor groove of DNA and have been shown to inhibit many protein-DNA complexes. However, some major groove-binding proteins can co-occupy the same DNA sequences as polyamides. Presented here are polyamide-intercalator conjugates that specifically bind target regions of DNA and deliver a non-specific intercalator to an adjacent site. The studies detail intercalative unwinding of specific DNA sequences to allosterically inhibit any protein:DNA complex. The evolution of sequence-specific polyamides to bisintercalate DNA and cause larger distortion of the helix is described. The success of hybrid molecules containing mixed DNA binding modes led to the development of a bis-polyamide-intercalator motif, modeled after the natural product actinomycin D, which is capable of specifically binding extended sequences of DNA. Also described is a polyamide-intercalator series which shows large fluorescence enhancement upon specific DNA binding and may be useful in detecting specific DNA sequences within living cells.", "doi": "10.7907/d0v0-pb71", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1999", "collection": "thesis", "collection_id": "1999", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05242005-143543", "primary_object_url": { "basename": "SS_Preface.pdf", "content": "final", "filesize": 140358, "license": "other", "mime_type": "application/pdf", "url": "/1999/7/SS_Preface.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Prediction of Structure and Antagonist Binding Site in Human and Rodent Chemokine Receptor 1", "author": [ { "family_name": "Sharma", "given_name": "Shantanu", "clpid": "Sharma-Shantanu" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chemoattractant cytokines (chemokines) are small proteins that are known to play a key role in the development of numerous autoimmune and inflammatory diseases. The signal transduction cascade responsible for this pathology is initiated by chemokine binding to a G-protein coupled receptor (GPCR). Since therapeutic intervention would involve inhibition of ligand binding, it follows that detailed understanding of the structures and binding sites of these receptors would lead to the rational design of such drugs. However, GPCRs are a class of integral membrane proteins whose structures are extremely difficult to determine via the conventional method of X-ray crystallography. Additionally, homology models based on the crystal structure of bovine rhodopsin (BR) have offered little structural insight into the remotely homologous chemokine receptors. In light of this information, our laboratory has developed a novel computational approach to predicting the structures and ligand binding sites of GPCRs with no information from the atomic coordinates of the crystal structure of BR.
\r\n\r\nIn this thesis we describe the use of the MembStruk procedure to predict the structure of human, mouse, and rat chemokine receptor 1 (CCR1). Interhelical interactions that stabilize the conformation of each receptor are discussed in detail, and where appropriate comparisons are made to information gleaned from the crystal structure of BR. The side chain placements of conserved residues are found to be different across the human and rodent species, accounting for binding differentials not previously explained by homology models. To improve the binding of a low affinity small molecule antagonist, point mutation candidates in human CCR1 are predicted.
\r\n\r\nValidation of the human CCR1 structure is achieved through prediction of the antagonist binding site, to which a series of known antagonists are docked and scored for comparison to experimental structure-activity data. The ligand binding energies are in excellent agreement with the experimentally known trend in binding affinities, and results from a virtual ligand screening calculation (Dr. Sabine Schlyer, Berlex/Schering AG) also support the validity of the structural model. This work in this thesis provides the basis for the design of receptor-specific antagonists to human and rodent CCR1, thus accelerating the drug discovery process.
", "doi": "10.7907/j4zd-ny21", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1835", "collection": "thesis", "collection_id": "1835", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05162005-153745", "primary_object_url": { "basename": "SLWidicusWeaver_thesis.pdf", "content": "final", "filesize": 3693636, "license": "other", "mime_type": "application/pdf", "url": "/1835/25/SLWidicusWeaver_thesis.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Rotational Spectroscopy and Observational Astronomy of Prebiotic Molecules", "author": [ { "family_name": "Widicus Weaver", "given_name": "Susanna Leigh", "orcid": "0000-0001-6015-3429", "clpid": "WidicusWeaver-Susanna-Leigh" } ], "thesis_advisor": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Collier", "given_name": "C. Patrick", "orcid": "0000-0002-8198-793X", "clpid": "Collier-C-P" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "local_group": [ { "literal": "Caltech Submillimeter Observatory" }, { "literal": "Astronomy Department" }, { "literal": "div_chem" } ], "abstract": "It is now widely believed that prebiotic molecules were delivered to the early Earth by planetesimals and their associated interplanetary dust particles. Yet the formation pathways for these molecules are not clear. Amino acids and sugars have been found in carbonaceous chondrites, but only much simpler species have been detected in the interstellar medium (ISM). Prebiotic organics could have formed in the ISM and been directly incorporated into planetesimals, or simpler species could have formed in the ISM and then been incorporated into planetesimals, undergone further processing, and been delivered to Earth. Limits on interstellar chemistry must therefore be established through observational astronomy before potential prebiotic formation pathways can be assessed. These observations require laboratory spectroscopic investigation of the species of interest.
\r\n\r\nThis thesis is an interdisciplinary study involving laboratory rotational spectroscopy and astronomical observations of several key prebiotic molecules. The laboratory work has focused on obtaining the rotational spectra of the simplest three-carbon ketose sugar, 1,3-dihydroxyacetone, and its structural isomers methyl glycolate and dimethyl carbonate, as well as aminoethanol, the predicted interstellar precursor to alanine. The pure rotational spectral analysis of the low-lying torsional states of the simplest alpha-hydroxy aldehyde, glycolaldehyde, has also been completed. The original Balle-Flygare Fourier transform microwave spectrometer was used to obtain the microwave spectra, while both the Jet Propulsion Laboratory and Caltech direct absorption flow cell spectrometers were used for additional direct absorption millimeter and submillimeter studies.
\r\n\r\nThe results of these laboratory experiments were used to guide observational searches with the Caltech Submillimeter Observatory, the Owens Valley Millimeter Array, and the Green Bank Telescope toward the hot core sources Sgr B2(N-LMH), Orion Hot Core/Compact Ridge, and W51 e1/e2. Evidence has been found for the presence of dihydroxyacetone and methyl glycolate in Sgr B2(N-LMH).
\r\n\r\nThese results have important implications for interstellar grain surface chemistry, and proposed additions to grain surface chemical models are also discussed. Reactions involving surface radicals and molecules containing carbonyl groups can efficiently compete with the simple grain surface reactions included in previous models. Such aldehyde abstraction reactions should be considered as pathways to complex carbonyl-containing species on interstellar grain surfaces.
", "doi": "10.7907/CN1R-H437", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2303", "collection": "thesis", "collection_id": "2303", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302005-223153", "primary_object_url": { "basename": "gh_thesis_5_30_05.pdf", "content": "final", "filesize": 7711441, "license": "other", "mime_type": "application/pdf", "url": "/2303/1/gh_thesis_5_30_05.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Advances in Computational Protein Design: Development of More Efficient Search Algorithms and their Application to the Full-Sequence Design of Larger Proteins", "author": [ { "family_name": "Hom", "given_name": "Geoffrey Kai Tong", "clpid": "Hom-Geoffrey-Kai-Tong" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Pierce", "given_name": "Niles A.", "orcid": "0000-0003-2367-4406", "clpid": "Pierce-N-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Protein design is the art of choosing an amino acid sequence that will fold into a desired structure. Computational protein design aims to quantify and automate this process. In computational protein design, various metrics may be used to calculate an energy score for a sequence with respect to a desired protein structure. An ongoing challenge is to find the lowest-energy sequences from amongst the vast multitude of sequence possibilities. A variety of exact and approximate algorithms may be used in this search.
\r\n\r\nThe work in this thesis focuses on the development and testing of four search algorithms. The first algorithm, HERO, is an exact algorithm, meaning that it will always find the lowest-energy sequence if the algorithm converges. We show that HERO is faster than other exact algorithms and converges on some previously intractable designs. The second algorithm, Vegas, is an approximate algorithm, meaning that it may not find the lowest-energy sequence. We show that, under certain conditions, Vegas finds the lowest-energy sequence in less time than HERO. The third algorithm, Monte Carlo, is an approximate algorithm that had been developed previously. We tested whether Monte Carlo was thorough enough to do a challenging computational design: the full-sequence design of a protein. Monte Carlo didn\u2019t find the lowest-energy sequence, although a similar sequence from Vegas folded into the desired structure. Several biophysical methods suggested that the Monte Carlo sequence should also fold into the desired structure. Nevertheless, the Monte Carlo structure as determined by X-ray crystallography was markedly different from the predicted structure. We attribute this discrepancy to the presence of a high concentration of dioxane in the crystallization conditions. The fourth algorithm, FC_FASTER, is an approximate algorithm for designs of fixed amino acid composition. Such designs may accelerate improvements to the physical model. We show that FC_FASTER finds lower-energy sequences and is faster than our current fixed-composition algorithm.
", "doi": "10.7907/M4R9-YM51", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1749", "collection": "thesis", "collection_id": "1749", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05122005-083103", "primary_object_url": { "basename": "Full_thesis.pdf", "content": "final", "filesize": 45442996, "license": "other", "mime_type": "application/pdf", "url": "/1749/12/Full_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structural Dynamics of Complex Molecules by Ultrafast Electron Diffraction : Concepts, Methodology and Applications", "author": [ { "family_name": "Srinivasan", "given_name": "Ramesh", "clpid": "Srinivasan-Ramesh" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "thesis_committee": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The central theme in ultrafast electron diffraction (UED) is the elucidation of the structural dynamics of transient molecular entities. With properly timed sequences of ultrafast electron pulses, it is now possible to image complex molecular structures in the four dimensions of space and time with resolutions approaching 0.01 [angstroms] and 1 ps, respectively. Reaching this spatiotemporal resolution on the atomic scale has been the driving force behind the development and application of the third generation UED instrument\u2013-the subject of this dissertation. The current state-of-the-art in resolutions and sensitivity, together with theoretical advances, has made possible the direct determination of transient structures, leading to studies of diverse molecular phenomena hitherto not accessible to other techniques. By freezing structures on the ultrafast timescale, we are able to develop concepts that correlate structure with dynamics. Examples include structure-driven radiationless processes, dynamics-driven reaction stereochemistry, and non-equilibrium structures exhibiting negative temperature, bifurcation, or selective energy localization in bonds. These successes in the studies of complex molecular systems, even without heavy atoms, establish UED as a powerful method for mapping out temporally changing molecular structures in chemistry, and potentially, in biology.", "doi": "10.7907/DTG2-5932", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2084", "collection": "thesis", "collection_id": "2084", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262004-173612", "primary_object_url": { "basename": "Giannetti_dissertation.pdf", "content": "final", "filesize": 31215100, "license": "other", "mime_type": "application/pdf", "url": "/2084/1/Giannetti_dissertation.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Biochemical, Biophysical, and Cellular Investigations of the Interactions of Transferrin Receptor with Transferrin and the Hereditary Hemochromatosis Protein, HFE", "author": [ { "family_name": "Giannetti", "given_name": "Anthony Michael", "clpid": "Giannetti-Anthony-Michael" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Hereditary hemochromatosis (HH) is a prevalent genetic disorder that results in the daily excess absorption of dietary iron. If untreated this disease leads to systemic organ failure and death. HH is caused by mutations to the gene coding for a protein called HFE, a type I transmembrane glycoprotein with a demonstrated role in regulating cellular iron homeostasis. HFE binds to the cell-surface receptor transferrin receptor (TfR), a dimeric type II transmembrane glycoprotein responsible for iron uptake into most mammalian cell types. TfR binds iron-loaded transferrin (Fe-Tf) from the blood and transports it to acidic recycling endosomes where iron is released from Fe-Tf in a TfR-facilitated process. Iron-free transferrin (apo-Tf) remains bound to TfR and is recycled to the cell surface, where apo-Tf rapidly dissociates from TfR upon exposure to the basic pH of blood. HFE and Fe-Tf can bind simultaneously to TfR to form a ternary complex, but HFE binding to TfR lowers the apparent affinity of the Fe-Tf/TfR interaction. This reduction could result from direct competition between HFE and Fe-Tf for receptor binding sites, from negative cooperativity, or both. We sought to understand the mechanism of HFE, Fe-Tf, and apo-Tf binding by TfR to help define HFE's role in iron homeostasis. We determined the binding constants for HFE, Fe-Tf, and apo-Tf to an extensive set of site-directed TfR mutants and discovered that HFE and Tf bind to an overlapping site on TfR, indicating the two proteins compete with each other for receptor binding. The mutagenesis results also identified differences in the contact points between TfR and the two forms of Tf, Fe-Tf and apo-Tf. By combining the mutations that are required for apo-Tf, but not Fe-Tf, binding we find that a highly conserved hydrophobic patch on the TfR surface is required for the receptor-mediated stimulation of iron release from Fe-Tf. From these data we propose a structure-based model for the mechanism of TfR-assisted iron release.
\r\n\r\nTo explore the mechanism of the HFE-induced affinity reduction for Fe-Tf binding by TfR, we engineered a heterodimeric TfR (hdTfR) that contains mutations such that one TfR chain binds only HFE and the other binds only Fe-Tf. Competition binding experiments using hdTfR demonstrate that TfR does not exhibit cooperativity in heterotropic ligand binding, suggesting that some or all of HFE's effects on iron homeostasis result from competition with Fe-Tf for TfR binding. Using transfected cell lines we show that HFE is dependent on its interactions with TfR for transport to endosomal compartments and that competition with extracellular Fe-Tf can alter HFE trafficking patterns. These data suggest that HFE's role in iron homeostasis is as a sensor of body iron status.
", "doi": "10.7907/06QJ-JW93", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2354", "collection": "thesis", "collection_id": "2354", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-214823", "primary_object_url": { "basename": "Thesis_AllPages.pdf", "content": "final", "filesize": 7844325, "license": "other", "mime_type": "application/pdf", "url": "/2354/1/Thesis_AllPages.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Computational Design and Experimental Characterization of Protein Oligomers", "author": [ { "family_name": "Huang", "given_name": "Po-Ssu", "orcid": "0000-0002-7948-2895", "clpid": "Huang-Po-Ssu" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Previous efforts in designing protein binding interfaces have focused on altering binding specificities. These methods fall short, however, when applied to the design of novel binding sites due to difficulties in accurately modeling protein backbones. The goal of this project is to create dimers from monomeric proteins. We developed a special docking algorithm that positions the member protein subunits to a plausible configuration with respect to each other using parameters determined from known complex structures. The docking procedure treats the proteins as rigid bodies and uses Fourier correlation theorem and fast Fourier transform to efficiently search for dimers with the highest interfacial surface complementarities. Using the docked structures as scaffolds for design and employing hydrophobic surface residues to drive dimer formation, we have demonstrated two successful designs, one heterodimer and one homodimer, using protein G and engrailed homeodomain respectively as the starting monomeric proteins. The designed dimers were characterized using circular dichroism, nuclear magnetic resonance, analytical ultracentrifugation, and X-ray crystallography methods. This is the first report of computationally designed de novo protein homodimers generated using a combination of protein docking and protein design tools. These results suggest that this strategy can be used to address the protein recognition problem, and is generally applicable to creating novel binding sites with compatible binding partners.", "doi": "10.7907/3DZW-2R54", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2350", "collection": "thesis", "collection_id": "2350", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-144201", "primary_object_url": { "basename": "HansenPC.pdf", "content": "final", "filesize": 13507896, "license": "other", "mime_type": "application/pdf", "url": "/2350/1/HansenPC.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Microfluidic Technologies for Structural Biology", "author": [ { "family_name": "Hansen", "given_name": "Carl Lars Genghis", "clpid": "Hansen-Carl-Lars-Genghis" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Painter", "given_name": "Oskar J.", "orcid": "0000-0002-1581-9209", "clpid": "Painter-O" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "In the post-genomic era, X-ray crystallography has emerged as the workhorse of large-scale structural biology initiatives that seek to understand protein function and interaction at the atomic scale. Despite impressive technological advances in X-ray sources, phasing techniques, and computing power, the determination of protein structure has been severely hampered by the difficulties in obtaining high-quality protein crystals. Emergent technologies utilizing microfluidics now have the potential to solve these problems on several levels, both by allowing researchers to conduct efficient assays in nanoliter reaction volumes, and by exploiting the properties of mass-transport at the micron scale to improve the crystallization process. The technique of Multilayer Soft Lithography (MSL) has been used to developed a set of microfluidic tools suitable for all stages of protein crystallogenesis, including protein solubility phase-space mapping, crystallization screening, harvesting, and in silicone diffraction studies. These tools represent the state of the art in on-chip fluid handling functionality and have been demonstrated to dramatically improve protein crystallization.\r\n", "doi": "10.7907/N9T3-7114", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1599", "collection": "thesis", "collection_id": "1599", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05042004-203854", "primary_object_url": { "basename": "Preface.pdf", "content": "final", "filesize": 277016, "license": "other", "mime_type": "application/pdf", "url": "/1599/8/Preface.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structure and Function Studies of the Human Dopamine Receptors", "author": [ { "family_name": "Kalani", "given_name": "M. Yashar S.", "orcid": "0000-0002-5923-1255", "clpid": "Kalani-M-Yashar-S" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Vaidehi", "given_name": "Nagarajan", "clpid": "Vaidehi-N" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Dopamine neurotransmitter and its receptors play a critical role in cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's and schizophrenia would be significantly enhanced with the availability of the three-dimensional (3-D) structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. In this thesis, I report the 3-D structures of the 5 subtypes of the human dopamine receptors, predicted from primary sequence using first principles theoretical and computational techniques. I use the term \"first principles\" to mean that we do not use the high resolution crystal structure of rhodopsin as a template, nor do we use homology modeling or threading of any kind to determine the structure. Predicting the binding sites, and the relative binding affinities of endogenous ligands and various pharmaceuticals to the 5 receptors validates the predicted structures. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies and give relative binding affinities that correlate well with experiment. The predicted binding site for dopamine and agonists is located between transmembrane helices (TM) 3, 4, 5, and 6, while the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM 5. We identify characteristic differences between the binding sites of agonists and antagonists, as well as factors that cause differential binding to the 5 subtypes of the human dopamine receptors.
\r\n\r\nThis thesis consists of five chapters that have, or will shortly result in publications. The first chapter is a brief introduction to the field, the motivation for the project, my scientific contributions, and contribution of others on the team. Chapter two introduces the methods and their successes at reproducing experimentally known results for the human D2 dopamine receptor; it discusses, in great detail, the active site of pharmaceutical agonists and antagonists to the human D2 dopamine receptor, and highlights the strengths and shortcomings of homology modeling for membrane bound proteins; this chapter will be submitted for publication to the Journal of Molecular Biology. Chapter three reports the results of a blind study performed in collaboration with Aventis Pharmaceuticals. For this study, we were provided with the two-dimensional structure of 9 antagonists and were asked to predict their binding sites, binding affinities, and to explain the differential binding of the ligands to the human D2 and D3 dopamine receptors and the human a1A adrenergic receptor. The results of this study are in preparation for submission to the Journal of Medicinal Chemistry. Chapters four and five of the thesis give preliminary results of comparative studies of the agonist and antagonist binding sites of the five subtypes of the human dopamine receptors. Chapter 6 contains results of another blind study on the G2A receptor with Professor Owen Witte.
\r\n\r\nIn addition to the six main chapters, this thesis contains 6 independent appendices that report results of similar studies in other systems. The first 2 appendices are work that has already been published. The remaining 4 appendices will shortly result in publications, but at this time, they are not publication worthy; these appendices represent data that has been analyzed but has not been written in paper format.
\r\n\r\nIn addition, I would like to make note of the studies that I have conducted on the 9 subtypes of the human adrenergic receptors with Mr. Peter Freddolino, the 4 human histamine receptors that were conducted with Mr. Freddolino and Mr. Maziyar Kalani, and the 4 G2A-like lipid receptors conducted with Mr. Rene Trabanino, Dr. Radu, Dr. Yang, and Professor Owen Witte of the Howard Hughes Medical Institute at the David Geffen School of Medicine at the University of California, Los Angeles.
", "doi": "10.7907/T6NV-7W30", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1856", "collection": "thesis", "collection_id": "1856", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05182004-181406", "primary_object_url": { "basename": "TitlePage.pdf", "content": "final", "filesize": 48514, "license": "other", "mime_type": "application/pdf", "url": "/1856/2/TitlePage.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Oxidative DNA Damage by Long-Range Charge Transport", "author": [ { "family_name": "Delaney", "given_name": "Sarah", "orcid": "0000-0002-8366-3808", "clpid": "Delaney-Sarah" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ever since the elucidation of the double helical structure of DNA, it has been proposed that the stack of base pairs within the double helix may mediate charge transport (CT) reactions. In fact, CT through DNA can result in chemistry at a distance, yielding oxidative DNA damage at a site remote from the bound oxidant. DNA CT chemistry depends upon coupling within the stacked base pair array, and this chemistry is remarkably sensitive to sequence-dependent DNA structure and dynamics. Using a variety of octahedral transition metal complexes, DNA CT has been probed to explore mechanistic considerations and biological possibilities.
\r\n\r\nInteractions with DNA by a family of ruthenium(II) complexes bearing the dipyridophenazine (dppz) ligand or its derivatives have been examined. An intercalative binding mode has been established based on luminescence enhancements in the presence of DNA, excited state quenching, fluorescence polarization values and enantioselectivity. Oxidative damage to DNA by these complexes using the flash/quench method has also been examined. A direct correlation between the amount of guanine oxidation obtained via DNA CT and the strength of intercalative binding was observed. These results support the importance of close association and intercalation for DNA-mediated CT. Electronic access to the DNA base pairs, provided by intercalation of the oxidant, is a prerequisite for efficient CT through the DNA pi-stack.
\r\n\r\nUsing polypyridyl ruthenium complexes, a reductive flash/quench scheme in DNA has also been explored. The flash/quench scheme previously utilized in DNA studies involves an oxidative quencher and allows for examination of electronic hole transport through DNA. In contrast, a reductive flash/quench technique would allow for direct observation of electron transport through the base stack. In our studies, p-methoxydimethylaniline and potassium iodide have proven to be effective reductive quenchers of dipyridophenazine complexes of ruthenium. However, by transient absorption spectroscopy, high performance liquid chromatography, gel electrophoresis, and electron paramagnetic resonance we are unable to observe any DNA reduction products with the ruthenium complexes examined. Rates of back electron transfer may in fact be faster than trapping of the anion radical, thus hindering observation of long-range damage.
\r\n\r\nThe oxidative flash/quench technique was applied in probing DNA CT in a range of DNA assemblies containing a tethered ruthenium intercalator and methylindole (M), a low potential nucleobase analog, where radical formation at a distance as a function of DNA sequence could be examined both by laser spectroscopy and biochemical methods. Hole injection and subsequent formation of the methylindole radical cation were observed at a distance of over 30 \u0160at rates > 10e7 s-1 in assemblies containing no guanine bases intervening the ruthenium intercalator and GMG oxidation site. Radical yield was, however, strikingly sensitive to an intervening base mismatch; no significant methylindole radical formation was evident with an intervening AA mismatch. Also critical is the sequence at the injection site; this sequence determines initial hole localization and hence the probability of hole propagation. With guanine rather than inosine near the site of hole injection, decreased yields of radicals and long-range oxidative damage are observed. The presence of the low energy guanine site in this case serves to localize the hole and increase the probability of back reaction at the injection site therefore diminishing CT through the base pair stack.
\r\n\r\nDNA assemblies containing a pendant dppz complex of Ru(II) along with two oxidative traps, a site containing the nucleoside analog methylindole (5?-GMG-3?) and a 5?-GGG-3? site, were constructed to explore charge equilibration across the base pair stack. In these assemblies the base radicals form with a rate of 10e7 s-1. Interestingly, the rate of base radical formation does not change upon the addition of a second radical trap, the 5?-GGG-3? site; however the yield of methylindole oxidation is significantly lower. This observation indicates that the 5?-GGG-3? site is effective in competing for the migrating charge and provides a second trapping site. Importantly, switching the orientation of the two trapping sites does not affect the yield of oxidized products at either site. Therefore, in DNA both forward and reverse charge transport occur so as to provide equilibration across the duplex on a time scale that is fast compared to trapping at a particular site. Further evidence of charge equilibration results from incorporating an intervening base-stacking perturbation and monitoring the fate of the injected charge. These experiments underscore the dynamic nature of DNA charge transport and reveal the importance of considering radical propagation in both directions along the DNA duplex.
\r\n\r\nDNA conjugates containing adjacent duplex and guanine quadruplex assemblies have been designed to explore CT into quadruplex architectures. The quadruplex assemblies have been characterized structurally using circular dichroism and by assaying for chemical protection. Using an intercalating rhodium photooxidant, noncovalently bound or tethered to the duplex end, oxidizing radicals are found to be trapped in the folded quadruplex. Damage is observed almost exclusively at the external tetrads of the quadruplex. Little damage of the center tetrad is observed, due most likely to lowered efficiency of radical trapping within the quadruplex core. This pattern of damage is distinct from that observed for repetitive G sequences within duplex DNA. The data indicate, furthermore, that in the conjugates examined, the guanine quadruplex provides a more effective trap than a 5?-GG-3? guanine doublet within duplex DNA. Additionally, within these assemblies, sufficient base-base overlap must exist at the duplex/quadruplex junction to allow for charge migration. This funneling of damage to the quadruplex, as well as the unique pattern of damage within the quadruplex, requires consideration with respect to the analysis of oxidative DNA damage within the cell.
", "doi": "10.7907/9Q0X-TZ17", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1893", "collection": "thesis", "collection_id": "1893", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05202004-174324", "primary_object_url": { "basename": "ALL_THESIS_2.pdf", "content": "final", "filesize": 8738128, "license": "other", "mime_type": "application/pdf", "url": "/1893/1/ALL_THESIS_2.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Prediction of Structure, Function, and Spectroscopic Properties of G-Protein-Coupled Receptors: Methods and Applications", "author": [ { "family_name": "Trabanino", "given_name": "Rene Jouvanni", "clpid": "Trabanino-Rene-Jouvanni" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "G-protein-coupled receptors are of great pharmaceutical interest, comprising the majority of targets for currently marketed drugs. The theme of my thesis is the development of the structure prediction method, MembStruk, for the superfamily of G-protein-coupled receptors. The first part of this thesis focuses on the methods and their validation. There are several steps involved in MembStruk that are detailed and tested for membrane proteins with known structures in the first few chapters (Chapters 2-6). Specifically, the first principles methods for predicting the transmembrane helical ranges and the helix hydrophobic centers are tested. The program for predicting the transmembrane helical ranges, TM2ndS, ranks in the top two when comparing performance with other top prediction methods. And because it is based on general principles, it can be applied robustly for membrane protein families for which little structural information is available. The simulation of the EC-II closing is also tested on bovine rhodopsin. The use of the MembStruk method on bovine rhodopsin as a validation case is presented in detail (Chapter 2). The large majority (71%) of the residues involved in binding in rhodopsin are predicted and the protein structure itself is 2.84 \u00c5 coordinate root mean square error in the transmembrane main chain atoms from the crystal structure.
\r\n\r\nThe second part of the thesis discusses applications on various G-protein-coupled receptor systems. The application of the MembStruk method to other peptide chemokine G-protein-coupled receptors like CCR1 and CCR5 is discussed in Chapter 9. The fundamental scientific problems of G-protein-coupled receptor modulation of absorption and relaxation properties of a bound chromophore (retinal) are addressed and results are presented for the predictions of these properties.
\r\n\r\nThe prediction of structure and function of G-protein-coupled receptors would allow for structure-based drug design and a rational approach to reducing drug cross-reactivity across receptor families.
", "doi": "10.7907/VHED-4063", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1912", "collection": "thesis", "collection_id": "1912", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05212004-144036", "primary_object_url": { "basename": "Elmore_Thesis.pdf", "content": "final", "filesize": 16831479, "license": "other", "mime_type": "application/pdf", "url": "/1912/8/Elmore_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Investigations of Ion Channel Structure-Function Relationships Using Molecular Modeling and Experimental Biochemistry", "author": [ { "family_name": "Elmore", "given_name": "Donald Eugene, Jr.", "orcid": "0000-0002-8723-8710", "clpid": "Elmore-Donald-Eugene-Jr" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ion channels are integral membrane proteins found in all cells that mediate the selective passage of specific ions or molecules across a cell membrane. These channels are important in a diverse range of physiological processes, including signal transmission in the nervous system, sensory perception, and regulation of vital systems, such as circulation. This thesis discusses the use of computational chemistry methods, such as molecular dynamics (MD) and ab initio calculations, and experimental biochemical techniques, such as site-directed mutagenesis, in vivo bacterial assays, chemical cross-linking, and circular dichroism spectroscopy, in tandem to elucidate ion channel structure-function relationships. This research was catalyzed by the solving of atomic resolution crystal structures of the mechanosensitive channels of large and small conductance (MscL and MscS) by the Rees group. Although interesting themselves, these bacterial channels also provide good model systems for considering more complex eukaryotic channels.
\r\n\r\nMscL is an ion channel gated only by membrane tension. Initial studies of MscL verified the relevance of the crystal structure conformation under physiological conditions and compared different MscL homologues. Other work began to elucidate potentially unique structural and functional roles of the M. tuberculosis MscL C-terminal helical bundle. As well, interactions between the MscL channel protein and surrounding lipid and the potential relevance of helical kinking in MscL gating pathways were investigated. MscS is also gated by membrane tension, but its gating can be modulated by changes in transmembrane potential. Thus, studies on MscS began to identify the specific amino acid residues that are responsible for giving the channel its voltage sensitivity. Finally, computations predicting the conformation of nicotine in different solvent environments are discussed. Nicotine is a small molecule ligand that binds to and gates nicotinic acetylcholine receptors, and a thorough understanding of nicotine structure could aid efforts to elucidate receptor structure-function relationships and design new pharmaceuticals.
", "doi": "10.7907/47GW-HT46", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1868", "collection": "thesis", "collection_id": "1868", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05192004-092347", "primary_object_url": { "basename": "main.pdf", "content": "final", "filesize": 3096862, "license": "other", "mime_type": "application/pdf", "url": "/1868/1/main.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "M\u00f6ssbauer Diffractometry: Principles, Practice, and an Application to a Study of Chemical Order in \u2075\u2077Fe\u2083Al", "author": [ { "family_name": "Lin", "given_name": "Jiao", "clpid": "Lin-Jiao" } ], "thesis_advisor": [ { "family_name": "Fultz", "given_name": "Brent T.", "clpid": "Fultz-B-T" } ], "thesis_committee": [ { "family_name": "Fultz", "given_name": "Brent T.", "clpid": "Fultz-B-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Painter", "given_name": "Oskar J.", "clpid": "Painter-O" }, { "family_name": "Haile", "given_name": "Sossina M.", "clpid": "Haile-S-M" }, { "family_name": "Johnson", "given_name": "William Lewis", "clpid": "Johnson-W-L" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "For the first time, M\u00f6ssbauer powder diffractometry went beyond the proof-of-principal stage and was used to study unknown periodicities of defect-related chemical environments of Fe atoms in a partially-ordered \u2075\u2077Fe\u2083Al polycrystalline sample.
\r\n\r\nM\u00f6ssbauer powder diffractometry is based on two phenomena, the M\u00f6ssbauer effect and the Bragg diffraction. The M\u00f6ssbauer effect is sensitive to short-range order whereas diffractometry is sensitive to long-range order. Together, they enable M\u00f6ssbauer powder diffractometry to provide information on long-range periodicities of target atoms having specific short-range order.
\r\n\r\nBoth experimental and theoretical efforts are necessary for this novel technique to become practical. In this research, hardware and software for M\u00f6ssbauer powder diffractometry were improved. A kinematical diffraction theory for M\u00f6ssbauer powder diffractometry incorporating effects of interference between electronic and nuclear resonant scattering was developed. The applicability of the theory was verified by computer calculations that accounted for dynamical diffraction effects. A thorough analysis of polarization effects, including a polycrystalline average of polarization factors, was done systematically using spherical harmonic expansions.
\r\n\r\nMultiple diffraction patterns were measured at Doppler velocities across all nuclear resonances of \u2075\u2077Fe\u2083Al. On the basis of the theory developed, the superlattice diffractions were analyzed to provide data on the long-range order of Fe atoms having different numbers of Al neighbors. Comparing experimental data to calculations showed that Fe atoms having three Al atoms as first-nearest neighbors (1nn) have partial simple cubic long-range order, similar to that of Fe atoms with four Al 1nn. The simple cubic periodicity of Fe atoms with three Al 1nn was significantly lower than expected for homogeneous antisite disorder, however. Monte-Carlo simulations and transmission electron microscopy suggest that a significant fraction of aperiodic Fe atoms with three Al 1nn are near antiphase domain boundaries.
", "doi": "10.7907/JSZ7-BC77", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2295", "collection": "thesis", "collection_id": "2295", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302003-153624", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 3582227, "license": "other", "mime_type": "application/pdf", "url": "/2295/1/Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Amino Acid Radicals in Rhenium-Modified Copper Proteins", "author": [ { "family_name": "Wehbi", "given_name": "William Amine", "clpid": "Wehbi-William-Amine" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Oxidative flash quench of [Re(CO)3(phen)(His)]+ generates a high-potential [Re]2+ oxidant (E\u00b0 (Re2+/+) = 2 eV v. NHE), which has been used to obtain rates of electron transfer of Cu(I) oxidation in rhenium-modified azurins. These rates are enhanced over the [Ru(bpy)2(im)(His)]2+ analogues (E\u00b0 Ru3+/2+ ~ 1 eV), suggesting an alternate mechanism from driving force optimized, singe-step electron tunneling. To test whether other intermediates can be involved, oxidative freeze flash quench of the zinc(II) derivatives were undertaken. These experiments reveal that [Re]2+ can produce the amino acid radicals of tyrosine and cysteine, as detected by EPR. The properties of these radicals in structurally well-defined protein microenvironments in Pseudomonas aeruginosa azurin mutants have focused, in particular, on the g1 component of the g-tensor, which is sensitive to the strength of the hydrogen bond to the radical. The g1 for Tyr48 radical, which resides in a completely hydrophobic pocket and is inaccessible to solvent, is found to be greater than the g1 for the solvent exposed Tyr108 radical. This comparison could not be made for the cysteine radicals as Cys108 formed a sulfenyl radical upon oxidation; the Cys48 radical has been demonstrated to be a thiyl radical species and provides the EPR spectroscopic benchmark for a non-hydrogen bonded thiyl radical.
\r\n\r\nIn azurin mutants without any tyrosine, tryptophan, or cysteine residues, oxidative flash quench results in another organic based radical. This radical is located on the histidine imidazole ring that is coordinated to the rhenium atom. DFT calculations suggest that the spin density resides mainly on the imidazole ring when it is deprotonated. Corrected distances in the tunneling timetable to the imidazole ring from the copper atom predict an identical exponential decay in the electron transfer rates as for the ruthenium-labeled azurins. The rate enhancement is explained in terms of a \"trivial hop\" whereby Re2+ rapidly oxidizes the non-innocent histidine ligand in a proton dependent process; the histidine radical in turn oxidizes the copper atom or tyrosine, cysteine, or tryptophan when zinc is present. This model explains all of the enhanced Cu(I) oxidation rates by [Re]2+ and suggests that for Cu(I) oxidation in azurin, multistep electron tunneling through other amino acid radicals does not occur and that the observed radicals are generated in off-path processes.
\r\n", "doi": "10.7907/HF8D-1W16", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:245", "collection": "thesis", "collection_id": "245", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01202003-221429", "primary_object_url": { "basename": "BrandtThesis.pdf", "content": "final", "filesize": 9256488, "license": "other", "mime_type": "application/pdf", "url": "/245/1/BrandtThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Site-Specific Incorporation of Synthetic Amino Acids into Functioning Ion Channels", "author": [ { "family_name": "Brandt", "given_name": "Gabriel Shaw", "orcid": "0000-0002-9148-8042", "clpid": "Brandt-Gabriel-Shaw" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Synthetic amino acids may be introduced into functioning proteins by means of nonsense suppression, using tRNA aminoacylated with unnatural amino acids. This technique can be extended to living cells through micro-injection of mRNA and tRNA into Xenopus laevis oocytes. Introduction of synthetic amino acids into proteins has been used, broadly, for three purposes. First, sensitive probes have been incorporated into proteins, using side chain chemistry unavailable to naturally encoded amino acids. Second, reactive side chains have been developed which can drive conformational rearrangements of the protein. Third, natural post-translational modifications of protein side chains have been mimicked. The work presented here applies all of these approaches to the study of ion channels. A series of fluorinated Trp residues was incorporated into the nicotinic acetylcholine receptor (nAChR) to probe electrostatic effects on cation-pi mediated binding of nicotine and other agonists. Site-specific protein backbone cleavage of the nicotinic acetylcholine and purinergic P2X\u2082 receptors was undertaken, along with intersubunit photo-crosslinking in the nAChR. Caged tyrosine was employed to study tyrosine phosphorylation of an important modulatory site in the potassium channel Kir2.1. Finally, caged phosphoamino acid analogs were synthesized for further characterization of the effects of phosphorylation on ion channels.", "doi": "10.7907/3PYX-4P72", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:1322", "collection": "thesis", "collection_id": "1322", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04102003-004657", "primary_object_url": { "basename": "Pre_ch.pdf", "content": "final", "filesize": 169018, "license": "other", "mime_type": "application/pdf", "url": "/1322/7/Pre_ch.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mapping the Cytochrome C Folding Landscape", "author": [ { "family_name": "Lyubovitsky", "given_name": "Julia Gennadievna", "clpid": "Lyubovitsky-Julia-Gennadievna" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S. cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a timescale regulated by heme ligation events (~300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.
\r\n\r\nNonnative states of proteins are involved in a variety of cellular processes, including translocation of proteins across membranes and formation of amyloid fibrils. Probes that report on the structural heterogeneity of a polypeptide ensemble could resolve ambiguities in the classification of these states. We have shown that added anions shift the equilibrium between the compact and extended polypeptide structures that are present during refolding of Saccaromyces cerevisiae iso-1 cytochrome c. Specifically, at high salt concentrations (>= 700 mM), all the polypeptides are compact with a mean C-terminal fluorophore-heme separation quite close to that in the native protein (25 A). Addition of chemical detaturants, on the other hand, tends to shift the equilibrium towards unfolded structures.
\r\n\r\nFolding of modified Fe(II) cyt c was probed by fluorescence in presence of imidazole with NADH as photochemical sensitizer. At very high imidazole concentrations (400 mM), protein was still found to fold but the rate that coincides with Met80 ligation was slowed down significantly.
\r\n\r\nReductive flash-quench/scavenge experiments, in which ascorbic acid was used to scavenge MeODMAA+, were shown to keep ferrocyt c reduced for up to 500 ms. Electron injection into unfolded modified yeast Fe(III)cyt c was fast in comparison to injection using NADH as photochemical sensitizer. The overall electron transfer process was reversible. This photoreduction system could be used to trigger folding of Fe(II) cyt c to monitor the changes in dansyl fluorescence intensity on ms time scales.
", "doi": "10.7907/W2R9-Y195", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:4208", "collection": "thesis", "collection_id": "4208", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10222002-204928", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 2270596, "license": "other", "mime_type": "application/pdf", "url": "/4208/1/Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Ruthenium-Based Olefin Metathesis Catalysts Coordinated with NHeterocyclic Carbene Ligands: Synthesis and Applications", "author": [ { "family_name": "Morgan", "given_name": "John Philip", "clpid": "Morgan-John-Philip" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Imperiali", "given_name": "Barbara", "clpid": "Imperiali-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The improved synthesis and olefin metathesis activity of N-heterocyclic carbene (NHC)-coordinated ruthenium alkylidenes of the form (NHC)(L)x(Cl)2Ru=CHR (x = 1 or 2) are reported. In order to circumvent the handling of highly sensitive free carbenes, N-heterocyclic carbene \"adducts\" were prepared in high yields by the reaction of nucleophilic bases with N,N'-diarylimidazolium salts. Most notably, the addition of trichloromethyl anion to N,N'-dimesityl-4,5-dihydroimidazolium chloride produced an air-, moisture-, and temperature-stable crystalline adduct, 2-trichloromethyl-4,5-dihydro-imidazolidine. When this species is heated above the critical temperature of 55 degrees C in the presence of (PCy3)2(Cl)2Ru=CHPh, a single, clean phosphine substitution reaction occurs to form the NHC-coordinated benzylidene (NHC)(PCy)3(Cl)2Ru=CHPh in 84% isolated yield. This procedure has been successfully scaled up to industrial production and remains the most effective catalyst synthesis to date.
\r\n\r\nThe NHC-coordinated catalysts show dramatically expanded activity relative to their bis-phosphine counterparts. The high yielding, trans-stereoselective cross metathesis of various acroyl substrates is the first example of the ruthenium-catalyzed metathesis of olefins directly substituted with electron-withdrawing functionality. Ring-opening cross metathesis of acroyl species with relatively high ring strain cyclooctadiene and norbornene monomers has also been achieved in good yields and perfect regioselectivity when the norbornene is asymmetrically substituted with a bridgehead methyl group.
\r\n\r\nFurther expansion of the substrate scope was achieved when the catalyst's phosphine ligand was replaced with more weakly bound 3-bromopyridine (3-Br-pyr) ligands. The resulting catalyst (NHC)(3-Br-pyr)2(Cl)2Ru=CHPh produced synthetically useful yields (>= 67%) in the cross metathesis of acrylonitrile and terminal olefins (as opposed to less than 30% yield with the phosphine-coordinated catalyst). NHC-coordinated catalysts therefore allow both electron-rich and electron-poor olefins to undergo metathesis in the same pot, potentially leading to synthetically valuable products containing electronically differentiated olefins.
\r\n\r\nThe lower activity of phosphine-coordinated catalysts relative to those coordinated with 3-bromopyridine can be addressed by the addition of \"phosphine scavengers\" to the former. Higher pKa carboxylic acids (such as acetic and benzoic acids) are capable of accelerating catalysis as effectively as the much stronger hydrochloric acid, without concomitant catalyst decomposition. These properties make carboxylic acids the optimal choice for use with sensitive organic substrates.
\r\n", "doi": "10.7907/JFPH-1020", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:2126", "collection": "thesis", "collection_id": "2126", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272003-203637", "primary_object_url": { "basename": "Cheng_Thesis_PART_A.pdf", "content": "final", "filesize": 99809, "license": "other", "mime_type": "application/pdf", "url": "/2126/1/Cheng_Thesis_PART_A.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigations into the Enzymology and Biotechnology of the Hyperthermophilic Carboxypeptidase (PfuCP) from the Archaeon Pyrococcus furiosus", "author": [ { "family_name": "Cheng", "given_name": "Timothy Casey", "clpid": "Cheng-Timothy-Casey" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A novel metallocarboxypeptidase (PfuCP) from the hyperthermophilic archaeon Pyrococcus furiosus was purified and characterized to investigate its dependence on metal ion cofactors and to evaluate its suitability as a biotechnological tool for protein sequencing at elevated temperatures.
\r\n\r\nThe crystal structure reveals a dimer of primarily a-helical subunits that bears no resemblance to the \u03b1/\u03b2-hydrolase morphology of typical carboxypeptidases and which defines a new family of HEXXH metalloproteases (M32) based on primary sequence alignments. A deep active site groove appears to function not only in size-selection of substrates but also in modulating the activity and substrate affinity through complicated allosteric effects involving ambient ligands which may play a role in regulatory metabolism.
\r\n\r\nTwo forms of the enzyme were observed; one which retains stabilizing metal(s) that confer structural thermostability and a remarkable retention of activity to the dimer, and another demetallated form which has lost stability with regards to both dimeric integrity and activity. Difficulties in expressing a properly folded recombinant necessitated refolding of the expressed clone from inclusion bodies and further suggest that in vivo the stabilizing metal(s) may participate in folding a metastable enzyme.
\r\n\r\nThe apparent paradox of activation by only Co\u00b2\u207a and not Zn\u00b2\u207a is resolved into two issues, uncompetitive inhibition by the latter as seen in steady-state kinetic experiments, and intrinsic, electronic aspects of a catalytic Co\u00b2\u207a. Several explanations are proposed for the intrinsic rate enhancement of Co\u00b2\u207a over Zn\u00b2\u207a including the ability of Co\u00b2\u207a to modulate the potential energy surface for both reactants and transition states by virtue of its greater mobility within the protein framework.
\r\n\r\nThe broad amino acid specificity and rapid digestion by PfuCP in peptide sequencing trials show promise, and high-temperature protein sequencing has now been demonstrated for the first time.
", "doi": "10.7907/R8WE-R336", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:486", "collection": "thesis", "collection_id": "486", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02042003-160317", "primary_object_url": { "basename": "1-28-03_thesis.pdf", "content": "final", "filesize": 3461756, "license": "other", "mime_type": "application/pdf", "url": "/486/1/1-28-03_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Engineering Dioxygenases by Laboratory Evolution: A Comparison of Evolutionary Search Strategies", "author": [ { "family_name": "Joern", "given_name": "John Michael", "clpid": "Joern-John-Michael" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Gavalas", "given_name": "George R.", "orcid": "0000-0003-1468-6835", "clpid": "Gavalas-G-R" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Due to the unique and difficult chemistry they perform, the aromatic ring-hydroxylating dioxygenases are of interest as industrial catalysts. Unfortunately, an application-specific array of problems limits their utility. To address these problems through laboratory evolution, I developed methods for high-throughput screening of tens of thousands of dioxygenase variants. These methods rely on a phenol detection reagent (Gibbs reagent) and can be applied to liquid cultures or to growing bacterial colonies expressing variant enzymes.
\r\n\r\nRecombination of genes encoding homologous enzymes (\"family shuffling\") has emerged as a promising tool for evolutionary protein engineering. Using the dioxygenases as a model system, I have investigated the value of recombination as a search strategy for laboratory evolution. Chimeric dioxygenase libraries constructed by DNA shuffling are first evaluated for biases that limit sequence diversity using a probe hybridization approach in lieu of sequencing. This analysis shows that crossovers preferentially occur in regions with high sequence identity and that certain parent sequences can be preferred at particular gene positions.
\r\n\r\nHigh-throughput functional screening allowed characterization of substrate specificity for hundreds of dioxygenase chimeras. These data are coupled with sequence data to reveal sequence-function relationships and demonstrate the utility of recombination as a tool for functional genomics. One region of sequence is shown to be a primary determinant of substrate specificity for the enzymes studied. Furthermore, several sets of variant enzymes with similar functionality are shown to have sequence similarities.
\r\n\r\nRecombination and random mutagenesis are compared as search strategies for generating functionally-diverse dioxygenases. I screened similarly sized libraries of chimeric and mutant dioxygenases for variants with altered substrate specificity or activity toward n-hexylbenzene, which is not accepted by the parent enzymes. Both recombination and random mutagenesis gave rise to enzymes with altered substrate specificity, although such enzymes were more frequent in the chimeric libraries and more distinct specificities were found in the chimeric libraries. Only chimeras were active toward n-hexylbenzene. These results support the view that recombination is an effective search strategy for evolving substrate specificity, and may be more effective than random mutagenesis.
", "doi": "10.7907/61KV-P379", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:3169", "collection": "thesis", "collection_id": "3169", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08192002-161141", "primary_object_url": { "basename": "voigt_thesis.pdf", "content": "final", "filesize": 45686480, "license": "other", "mime_type": "application/pdf", "url": "/3169/1/voigt_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Computationally Optimizing the Directed Evolution of Proteins", "author": [ { "family_name": "Voigt", "given_name": "Christopher Ashby", "orcid": "0000-0003-0844-4776", "clpid": "Voigt-Christopher-Ashby" } ], "thesis_advisor": [ { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Fontana", "given_name": "Walter", "clpid": "Fontana-Walter" }, { "family_name": "Wang", "given_name": "Zhen-Gang", "orcid": "0000-0002-3361-6114", "clpid": "Wang-Zhen-Gang" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Directed evolution has proven a successful strategy for protein engineering. To accelerate the discovery process, we have developed several computational methods to optimize the mutant libraries by targeting specific residues for mutagenesis, and subunits for recombination. In achieving this goal, a statistical model was first used to study the dynamics of directed evolution as a search algorithm. These simulations improved our understanding of the relationship between parameters describing the search space (e.g., interactions between amino acids) and experimental search parameters (e.g., mutation rate and library size). Based on these simulations, a more detailed model was used to calculate the structural tolerance of each residue to amino acid substitutions. Further, a computational model was developed to optimize recombination experiments, based on the three-dimensional structure. Together, these computational techniques represent a major step towards information-driven combinatorial protein design. ", "doi": "10.7907/E4GF-EQ41", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:2299", "collection": "thesis", "collection_id": "2299", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05302003-172047", "primary_object_url": { "basename": "ardunn_thesis.pdf", "content": "final", "filesize": 13560094, "license": "other", "mime_type": "application/pdf", "url": "/2299/9/ardunn_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Sensitizer-Linked Substrates as Probes of Heme Enzyme Structure and Catalysis", "author": [ { "family_name": "Dunn", "given_name": "Alexander Robert", "orcid": "0000-0001-6096-4600", "clpid": "Dunn-Alexander-Robert" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ruthenium-diimine sensitizers (Ru-wires) with the structure [Ru(L2)L']2+, where L' is a perfluorobiphenyl bridge connecting 4,4'-dimethylbipyridine to the substrate adamantane or the heme ligand imidazole, bind to cytochrome P450cam with micromolar dissociation constants. Ru-wires can be used to trigger redox reactions on timescales faster than those achievable using conventional stopped-flow techniques: photoinduced heme reduction with an imidazole-terminated Ru-wire occurs in 40 ns. The large variation in ET rates among the Ru-diimine:P450 conjugates strongly supports a through-bonds model of Ru:heme electronic coupling.
\r\n\r\nThe Ru-wires also bind the murine inducible nitric oxide synthase (NOS) oxidase domain, both in the active site and to the hydrophobic surface patch that interacts with the NOS reductase domain. Rhenium-diimine probes with the structure [Re(4,7-dimethyl phenanthroline)(CO)3L]+, where L = imidazole-C12F8-imidazole (Re-im) or imidazole-C12F9 (Re-F9bp), bind in the NOS active site. Re-im (Kd = 6 nM) ligates the heme iron. Re-F9bp (Kd = 3.4 micromolar) produces a partial low- to high-spin conversion of the heme. Compounds with properties similar to the Ru- and Re-diimine probes may provide novel means of NOS inhibition.
\r\n\r\nLuminescent dansyl probes were designed to target cytochrome P450cam. D-4-Ad (dansyl-C4-adamantane) luminescence is quenched by Forster energy transfer upon binding (Kd = 0.83 micromolar), but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd ~ 0.02 micromolar) is not displaced from the enzyme even in the presence of a large excess of camphor. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.
\r\n \r\nCrystal structures of P450cam bound to ruthenium diimine and danysl probes reveal an open enzyme conformation that allows substrate access to the active center via a 22-A deep channel. Interactions of the probes with the channel illustrate the importance of exploiting protein dynamics in inhibitor design. Movements of the F, G and B' helices couple to conformational changes in active site residues implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that the structural flexibility of the F/G helix region allows the 54 human P450s to oxidize diverse substrates.
", "doi": "10.7907/RW1J-6665", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:6779", "collection": "thesis", "collection_id": "6779", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01252012-151300693", "primary_object_url": { "basename": "Belitsky_jm_2002.pdf", "content": "final", "filesize": 49802669, "license": "other", "mime_type": "application/pdf", "url": "/6779/1/Belitsky_jm_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA Binding Polyamides in Biological Systems", "author": [ { "family_name": "Belitsky", "given_name": "Jason Matthew", "clpid": "Belitsky-Jason-Matthew" } ], "thesis_advisor": [ { "family_name": "Janda", "given_name": "Kenneth C.", "clpid": "Janda-K-C" } ], "thesis_committee": [ { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Janda", "given_name": "Kenneth C.", "clpid": "Janda-K-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind selectively to a DNA sequence in the human genome are potentially useful tools for molecular biology and human medicine. Polyamides containing\r\nN-methylimidazole (Im) and N-methylpyrrole (Py) are small molecules that bind DNA according to a set of \"pairing rules\" with affinities and specificities that rival natural transcription factors. By directly competing with a given transcription factor or other DNA binding protein for its binding site, polyamide can cause inhibition of diverse biological processes, such as retroviral integration and gene transcription. Polyamides are presented which inhibit the in vitro integration activities for two retroviruses, M-MuLV and HIV-l. Polyamides are described that inhibit TBP binding to the HER2 promoter, a gene implicated in human breast cancer. Failure to achieve inhibition of HER2 transcription in cell culture led to the surprising discovery that polyamide-fluorescent dye conjugates are cell permeable, but that nuclear localization does not occur in many cell lines. Efforts toward modified polyamides with enhanced nuclear localization properties are presented. In order to extend the number of sequences amenable to high affinity recognition, alteration of the C-terminal polyamide tail has been investigated. The development of conditions for polyamide solid-phase synthesis on a new resin, which allows for the generation of \"truncated tail\" polyamides, is presented. During the original route to one of these compounds, an\r\nunexpected reaction was uncovered that leads to entirely different C-terminal tails.", "doi": "10.7907/WNV6-DH45", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6782", "collection": "thesis", "collection_id": "6782", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01262012-090632523", "primary_object_url": { "basename": "Copeland_kd_2002.pdf", "content": "final", "filesize": 75650200, "license": "other", "mime_type": "application/pdf", "url": "/6782/1/Copeland_kd_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Reactions of Metallointercalator-Peptide Conjugates and DNA", "author": [ { "family_name": "Copeland", "given_name": "Kimberly Davis", "clpid": "Copeland-Kimberly-Davis" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A family of metallointercalator-peptide conjugates for reaction with DNA has been constructed. In these chimeras, the metallointercalator provides binding affinity for\r\nDNA and the peptide contributes reactivity. With the goal of creating an artificial nuclease, we have tethered metal-binding peptides to a sequence neutral intercalator,\r\n[Rh(phi)_2bpy']^(3+) (phi = phenanthrenequinone diimine, bpy' = 4-butyric acid-4' -methyl-2,2'-bipyridine). This is a general strategy, and we have observed Zn^(2+)-promoted\r\ncleavage of plasmid DNA with widely different peptides: a designed helical peptide with histidine residues, and a hairpin peptide modeled after the active site of the BamHI\r\nendonuclease. To optimize the peptide composition of our artificial nuclease we created a library of 16,000 conjugates, but no new active conjugates were identified with this combinatorial strategy. To achieve oxidative cleavage of the DNA backbone we have also used our intercalator-peptide conjugates to deliver copper to DNA. Tethered peptides containing histidine residues promote oxidative strand scission in DNA restriction fragments and oligonucleotides in the presence of Cu^(2+) and a reducing agent. Importantly, by comparing the photocleavage pattern of the rhodium intercalator with the copper cleavage pattern of the metal-binding peptide, the interactions of the conjugate with DNA could be dissected. Finally, short peptides were tethered to [Ru(phen)(bpy')(dppz)]^(2+) \r\n(phen = 1,10-phenanthroline, dppz = dipyridophenazine) to\r\ncreate fluorescent DNA crosslinking agents. Through a flash-quench reaction, the ruthenium intercalator generates guanine radicals in a DNA duplex. These guanine\r\nradicals can react with water or oxygen, but also with tethered peptides to produce pennanent DNA-peptide crosslinks. The DNA-peptide crosslinks were detected by gel\r\nelectrophoresis and absorbance measurements, and characterized by mass spectrometry. Although they have low affinity for DNA, untethered peptides could also be crosslinked to DNA using the ruthenium chemistry. The peptide composition influences conjugate binding and the extent and pattern of crosslinking; indeed, positively charged residues were essential for effective crosslinking. Although the flexibility of our tethered peptides\r\nis an obstacle to the rational design of reactive conjugates, we have demonstrated that peptides can mediate a variety of reactions if delivered to DNA by metallointercalators.", "doi": "10.7907/MR9T-7W57", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6376", "collection": "thesis", "collection_id": "6376", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052011-134334164", "primary_object_url": { "basename": "Sundaresan_v_2002.pdf", "content": "final", "filesize": 78961721, "license": "other", "mime_type": "application/pdf", "url": "/6376/1/Sundaresan_v_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Selective Molecular Recognition in Imprinted Polymeric Adsorbents and in Biological Macromolecules", "author": [ { "family_name": "Sundaresan", "given_name": "Vidyasankar", "clpid": "Sundaresan-Vidyasankar" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes the synthesis and use of molecularly imprinted polymeric adsorbents for use in ligand-exchange chromatographic separations of structurally similar substrates. A general model of stereo selectivity is also described, which can be applied both to chromatographic adsorbents and to biological receptors.
\r\n\r\nCrosslinking polymerization of trimethylolpropane trimethacrylate (TRIM), under controlled conditions yields macroporous polymers bearing surface-accessible unpolymerized methacrylate residues. These residues have been utilized for copolymerization with different functional monomers to obtain composite polymer matrices with surface coatings of functional polymer chains. Surface modification has been carried out by molecular imprinting, using ternary Cu^(2+) complexes of [N-(4-vinylbenzyl)imino]diacetate and bisimidazole templates, with ethylene glycol dimethacrylate as comonomer. Selectivity characteristics similar to bulk-copolymerized polymers have been observed. The physicochemical characteristics of these functional polymer matrices have been evaluated by ^(13)C NMR, X-ray photoelectron spectroscopy, IR spectroscopy, and scanning electron microscopy.
\r\n\r\nThe ability of molecular imprinting to impart enantioselectivity to polymeric adsorbents has been studied using Cu^(2+) complexes of the achiral monomer [N-(4-vinylbenzyl)imino]diacetate and \u03b1-amino acids. Crosslinking polymerization with ethylene glycol dimethacrylate as the comonomer yields polymeric adsorbents capable of enantioresolutions of underivatized \u03b1-amino acids. Chromatographic adsorbents have been prepared by grafting the imprinted polymer on to silica particles. The observed enantioselectivity increases corresponding to the size of the side chain of the amino acid used as template, with the best enantioresolutions being obtained for materials imprinted against phenylalanine (~1.65 for D,L-phenylalanine enantioresolution). Adsorbents imprinted for alanine show negligible enantioselectivity. Cross-selectivity patterns towards non-template amino acids have been investigated, and the ability of an amino acid imprinted material to resolve analogous chiral amines has been demonstrated.
\r\n\r\nThe mechanisms underlying enantioselectivity in imprinted polymers are discussed in terms of the three-point interaction model. This model has been extended to a stereocenter-recognition (SR) model for substrates with multiple stereocenters. For N stereocenters in a linear chain, it has been demonstrated that a minimum of N + 2 interactions need to be distributed over all stereocenters, such that three effective interactions exist per stereocenter. The general applicability of the SR model is demonstrated for biological ligand-receptor interactions, by reinterpreting several previous experimental observations.
", "doi": "10.7907/jvev-x884", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6993", "collection": "thesis", "collection_id": "6993", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04302012-143643233", "primary_object_url": { "basename": "Marshall_sa_2002.pdf", "content": "final", "filesize": 12600744, "license": "other", "mime_type": "application/pdf", "url": "/6993/1/Marshall_sa_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Stability and Conformational Specificity in Protein Design: Models for Binary Patterning and Electrostatics", "author": [ { "family_name": "Marshall", "given_name": "Shannon Alicia", "clpid": "Marshall-Shannon-Alicia" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Binary patterning (the arrangement of hydrophobic and polar amino acids) and electrostatics are important determinants of the stability and conformational specificity of designed proteins. We have developed methods to to select the optimal binary pattern and model electrostatics in protein design studies. The Genclass method of binary patterning uses a solvent accessible surface generated from backbone coordinates of the target fold and \"generic\" side chains, constructs whose size and\r\nshape are similar to an average amino acid. Each position is classified according to the solvent exposure of its generic side chain. The method was tested by analyzing several proteins in the Protein Data Bank and by experimentally characterizing homeodomain variants whose binary patterns were systematically varied. Selection of the optimal binary pattern results in a designed protein that is monomeric, well-folded, and hyperthermophilic. Homeodomain variants with fewer hydrophobic residues are destabilized, additional hydrophobic residues induce aggregation. The optimal variant was further characterized by nuclear magnetic resonance spectroscopy. Binary patterning, in conjunction with a force field that models folded state energies, appears sufficient to satisfy two basic goals of protein design: stability and conformational specificity.
\r\n\r\nElectrostatic interactions are critical determinants of protein structure and function. Computational protein design algorithms typically use fast methods based on Coulomb's law to model electrostatic interactions. These methods fail to accurately account for desolvation and solvent screening, which strongly attenuate electrostatic interactions in proteins. Using the current force field, we designed a 25-fold mutant with moderate stability similar to the wild type protein. Incorporating two classes of electrostatic interactions using simple rules yielded a nine-fold mutant of the initial design that is over\r\n3 kcal mol^(-1) more stable. The simple electrostatic model used in the ORBIT force field is unable to predict the experimentally determined stabilities of the designed variants. Finite difference Poisson-Boltzmann (FDPB) methods have substantially better predictive power, but are far too slow for problems with high combinatorial complexity. We have developed new strategies for modeling electrostatics in\r\nprotein design problems that utilize one- and two-body decomposable FDPB methods. Computational results indicate that this method has the accuracy and speed required for design calculations.
\r\n", "doi": "10.7907/4BVY-VN75", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6996", "collection": "thesis", "collection_id": "6996", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05012012-101216772", "primary_object_url": { "basename": "Wang_ccc_2002.pdf", "content": "final", "filesize": 5350283, "license": "other", "mime_type": "application/pdf", "url": "/6996/1/Wang_ccc_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Sequence Specific Trapping of Topoisomerase I by Camptothecin Polyamide Conjugates", "author": [ { "family_name": "Wang", "given_name": "Clay Chia Chun", "clpid": "Wang-Clay-Chia-Chun" } ], "thesis_advisor": [ { "family_name": "Readhead", "given_name": "Anthony C. S.", "orcid": "0000-0001-9152-961X", "clpid": "Readhead-A-C-S" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Readhead", "given_name": "Anthony C. S.", "orcid": "0000-0001-9152-961X", "clpid": "Readhead-A-C-S" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Polyamides containing N-methylpyrrole (Py)N-methylimidazole (Im) and 3-hydroxy-1-methylpyrrole (Hp) are cell permeable small molecules that bind as antiparallel pairs in the minor groove of DNA according to a set of \"pairing rules\" with affinities and specificities for predetermined sequences comparable to DNA-binding proteins. Several of these ring pairings have only been demonstrated when placed internal in a hairpin and not for the terminal position in hairpin polyamides. Several series of eight ring hairpin polyamides with modification at the N-terminal position were synthesized and characterized. We observed that at the terminal position (i) the Py/Py pair functions similar to when placed internally preferring A\u2022T and T\u2022A base pairs over O\u2022C and C\u2022O, (ii) the Hp/Py pair could not distinguish between T\u2022A over A-T possibly due to rotation of the terminal Hp to form an intramolecular hydrogen bond between the 3-hydroxyl hydrogen and the carboxamide oxygen which would orient the key hydroxyl recognition element away from the minor groove. A new aromatic pair, 2-hydroxy-6-methoxybenzamidell-methylpyrrole was designed and shown to distinguish T\u2022A from A\u2022T base pairs and both from O\u2022C/C\u2022O and (iii) the Py/Im pair in the classic eight ring hairpin motif showed no preference for C\u2022O base pair possibly due to the mispositioning of the Im residue located at the C-terminal end of the four ring polyamide subunit. Targeting of C\u2022O was accomplished by replacing a pyrrole with a flexible \u03b2-alanine and setting the imidazole back in register.
\r\n\r\nPyrrole-imidazole polyamides that target DNA sequences in the promoter have been shown to inhibit transcription of specific genes in cell culture. When bound to coding region of genes, polyamides do not appear to inhibit gene expression. A possible solution is to design molecule capable of modifying DNA when bound to the coding region. A series of polyamide-camptothecin conjugates were designed to trap the enzyme Topoisomerase I and induce cleavage at predetermined DNA sites. Cleavage yields were shown to be dependent on linker length between the DNA binding polyamide\r\nand the Topo I trapping camptothecin unit with the camptothecin unit with the longest linker showing greatest cleavage yield of over 90%.
\r\n", "doi": "10.7907/sphn-ma74", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:2545", "collection": "thesis", "collection_id": "2545", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112002-135418", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 3311617, "license": "other", "mime_type": "application/pdf", "url": "/2545/1/thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Crystallographic Studies of Invasin, a Bacterial Adhesion Molecule from Yersinia pseudotuberculosis", "author": [ { "family_name": "Hamburger", "given_name": "Zsuzsa Andrea", "clpid": "Hamburger-Zsuzsa-Andrea" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Bacterial pathogens, such as Yersinia pseudotuberculosis, must bind and enter normally non-phagocytic cells to establish infection. The protein responsible for mediating uptake of the bacterium is a 986-residue outer membrane protein called invasin. Invasin binds to several members of the beta 1 integrin family, presumably activating a reorganization of the host cytoskeleton to form pseudopods that envelop the bacterium. Integrin binding has been localized to the extracellular region of invasin (Inv497) comprised by the COOH-terminal 497 residues. In order to gain insight into host cell entry by Yersinia pseudotuberculosis, we solved the 2.3 \u0160crystal structure of Inv497. The structure reveals five domains that form a 180 \u0160rod with structural similarities to tandem fibronectin-III domains. The integrin-binding surfaces of invasin and fibronectin include similarly located key residues, but in the context of different folds and surface shapes. The structures of invasin and fibronectin provide an example of convergent evolution, in which invasin presents an optimized surface for integrin binding compared with host substrates. We have also initiated structural analyses of the NH2-terminal ~500 residues of invasin, which are required for outer membrane localization and for presentation of the integrin-binding region of invasin. We expressed this region of invasin as inclusion bodies in E. coli, and refolded the protein in the presence of detergents. We have also obtained microcrystals of this membrane protein. Circular dichroism studies indicate that this region of invasin is composed of mainly beta-structure. As the transmembrane regions of outer membrane proteins of known structure are beta-barrels, this region of invasin is also presumed to fold into such a structure. Proteolysis experiments suggest that the NH2-terminal 70 amino acids of invasin may form a separate domain from the invasin transmembrane region, analogous to that found in another outer membrane protein, the sucrose-specific porin ScrY. Equilibrium sedimentation analytical ultracentrifugation studies indicate the protein is monomeric in solution. Black bilayer experiments suggest that this region of the protein does not contain a pore and thus plays the role of an outer membrane anchor for the presentation of the integrin-binding domain on the cell surface", "doi": "10.7907/NKAY-AB98", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6900", "collection": "thesis", "collection_id": "6900", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04102012-133622013", "primary_object_url": { "basename": "Wong-Foy_ag_2002.pdf", "content": "final", "filesize": 5217076, "license": "other", "mime_type": "application/pdf", "url": "/6900/1/Wong-Foy_ag_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Ancillary Ligand Effects in Zirconium(IV) Aminoborollide and Nitrogen Chelated Pt(II) Complexes", "author": [ { "family_name": "Wong-Foy", "given_name": "Antek Golangco", "clpid": "Wong-Foy-Antek-Golangco" } ], "thesis_advisor": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Labinger", "given_name": "Jay A.", "clpid": "Labinger-J-A" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The preparation of new chloro derivatives of pentamethylcyclopentadienyl aminoborollide complexes of Zr are described. Treatment of the dianions 1.9-1.12 with Cp*ZrCl_3 yields Cp*{\u03b7^5-C_4H_4BN(Si(CH_3)_3)_2}ZrCl\u2022LiCl (1.13), Cp*{\u03b7^5-C_4H_4BNC(CH_3)_3Si(CH_3)_3}ZrCl\u2022Li\u2022CI (1.14). |Cp*{\u03b7^5-C_4H_4BN(Et)CH_2CH_2Net_2}ZrCl_2Li]_2 (1.15), and Cp*{\u03b7^5-2,5-Ph_2C_4H_2BNMe_2}ZrCl\u2022LiCl (1.16). The electronic spectra of these complexes were measured and compared to the parent complex Cp*{\u03b7^5-C_4H_4BN(CHMe_2)_2}ZrCl\u2022LiCl (1.17a) in THF solvent. In general, as the substituents directly bonded to nitrogen increase in size, a blue shift of the low energy, aminoborollide to zirconium charge transfer band (LMCT), occurs. \u03bb_(max) decreases in the order 1.16 > 1.17a > 1.14 > 1.15 ~ 1.13. The anionic portions of complexes 1.13-1.16 have also been structurally characterized by x-ray crystallography. Although the changes are very small, in general a lengthened B-N bond correlates linearly with the observed blue shift of the LMCT band.
\r\n\r\nStudies directed towards the development of a Pt(II)-catalyzcd oxidation of ethylene to ethylene\r\nglycol based on the Shilov system for alkane functionalization is described. The first step is the activation of ethylene towards nucleophilic attack by water to generate a Pt(II) \u03b2-hydroxyalkyl intermediate that is oxidized in a second step to the Pt(IV) \u03b2-hydroxyalkyl. Reductive elimination via an S_N2-type mechanism at the \u03b1-C of the Pt(IV) \u03b2-hydroxyalkyl liberates the oxidized product leaving the reduced Pt(II) center to bind another equivalent of olefin. The first system examined was the methyl ethylene complex [(tmeda)PtMe(\u03b7^2-C_2H_4)][SbF_6,], 2.2. Nucleophilic attack at the bound ethylene was not observed: instead displacement of ethylene occurred. The bound ethylene in the neutral complexes cis-Cl_2PtL(\u03b7^2-C_2H_4), (L =PPh_3 (2.23), AsPh_3 (2.24), Me_2SO (2.25)) and trans-Cl_2Pt(\u03b7^2-C_2CH_4)(C_5H_5N). 2.26 arc susceptible towards attack by OH-. Under catalytic conditions (excess ethylene and H_2O_2) decomposition of 2.23, 2.24, and 2.25 was observed. In 2.26, 1 turnover was observed before decomposition occurred. The hound ethylene in the complex [(tmeda)PtCl(\u03b7^2-C_2H_4)][ClO_4] is activated towards nucleophilic attack by water and OH-, allowing the isolation of the Pt(II) \u03b2-hydroxyalkyl. This is rapidly oxidized to the Pt(IV) \u03b2-hydroxyalkyl by hydrogen peroxide. In the presence of HCl, it undergoes reductive elimination to yield 2-chlorocthanol\r\nand (tmeda)PtCl_2. Unfortunately, this system also showed no catalytic activity.
\r\n\r\nThe dicationic complexes (|(ArN=C(Me)-C(Me)=NAr)Pt(solv)_2 |X_2. (\u039br = 2.6-(CH_3)_2C_6,H_3: 3.5a:\r\nsolv = CH_3CN, X = CF_3SO_3-, BF_4-, SbF_6-; 3.5b: solv = (CH_3)_2CO, X = BF_4-, SbF_6-,) and 3.6\r\n[(CyN=C(H)-C(H)=NCy)Pt(CH_3CN)_2]X_2, (Cy = C_6H_(11) , X= OTf, BF_4-, Pf_6-, SbF_6-) were synthesized from the corresponding Pt dichlorides with 2 equiv. of AgX. The reaction of 3.5a with 1-phenylpyrazole, 2-phenylpyridine, 2-vinylpyridine, and 2-(2-thienyl)pyridine in acetone affords the cyclometalation products 3.11-3.14 via intramolecular C-H activation of an sp^2 C-H bond of the unsaturated sidegroup. Pyridines with saturated groups at the 2-position do not undergo a similar cyclomctalation reaction. 3.6 undergoes cyclometalation of one of the cyclohexyl groups, an example of sp^3 C-H bond activation. The later reaction proceeds only partway to completion, implying that an equilibrium has been reached: in the case where X = OTf, the equilibrium favors the starting dication. Furthermore, the intramolecular C-H activation occurs in trifluoroethanol but not in acetone under comparable conditions in contrast to the reactions of 3.5a with\r\nthe substituted pyridines.
\r\n\r\nThe diaqua complexe [(ArN=C(Me)-C(Me)=NAr)Pt(H_2O)_2]X_2, 4.3. (\u039br = 2.6-(CH_3)_2C_6H_3; X = OTf^-, BF_4^-) decompose in aqueous solution to yield a red-orange precipitate. Spectroscopic characterization of the precipitate by ^1H, IR, and conductivity measurements is consistent with C_(2v), symmetric structure containing hydroxo groups. Confirmation of the dicationic, dinuclear Pt(II) complex, 4.4, where the two Pt centers are bridged by two OH groups was revealed by x-ray crystallography.
", "doi": "10.7907/6xyc-e660", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:1686", "collection": "thesis", "collection_id": "1686", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05092002-155216", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 4044919, "license": "other", "mime_type": "application/pdf", "url": "/1686/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Characterization of the Mechanosensitive Channel of Large Conductance", "author": [ { "family_name": "Strop", "given_name": "Pavel", "clpid": "Strop-Pavel" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Osmoregulation is an essential process in bacteria and higher organisms regulated by the mechanosensitive ion channels. The mechanosensitive channel of large conductance (MscL) is an integral membrane protein that responds to pressure in an effort to prevent cell lysis during osmotic shock. Conversion of MscL from a membrane bound form to a water soluble form was attempted by three methods: computational design, random mutagenesis and chemical modification. The water soluble form of MscL was achieved with cysteine modification method. The stability, pH dependence, and C-terminal helix of MscL were also investigated.
\r\n \r\nThe structure of the cab beta-class carbonic anhydrase (Cab) has been determined to 2.1 A resolution. Cab exists as a dimer with a fold similar to plant beta-class carbonic anhydrases. The active site zinc is coordinated by Cys32, His87, and Cys90, with the tetrahedral coordination completed by a water molecule. The difference between plant and cab beta-class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes molecule near the active site, suggesting a proton transfer pathway to the solvent.
\r\n\r\nThe structure of the nitrogenase iron protein in the all-ferrous [4Fe-4S]0 form has been determined to 2.2 A resolution. The structure demonstrates that major conformational changes are not necessary to accommodate cluster reduction to the [4Fe-4S]0 state. A survey of [4Fe-4S] clusters coordinated by four cysteine ligands reveals that the [4Fe-4S] cluster of the iron protein has the largest accessible surface area, suggesting that solvent exposure may be relevant to the capability of existing in three oxidation states.
\r\n\r\nThe role of surface salt bridges in protein stabilization has been investigated. The NMR structure of a rubredoxin variant (PFRD-XC4) and the thermodynamic analysis of two surface salt bridges is presented here. The analysis shows that the surface sidechain to sidechain salt bridge between does not stabilize PFRD-XC4. The mainchain to sidechain salt bridge, however, stabilizes PFRD-XC4 by 1.5 kcal mol-1. The entropic cost of making a surface salt bridge involving the protein's backbone is reduced, since the backbone has already been immobilized upon protein folding.
\r\n", "doi": "10.7907/nkqb-gd95", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6797", "collection": "thesis", "collection_id": "6797", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312012-114925650", "primary_object_url": { "basename": "Nunez_me_2002.pdf", "content": "final", "filesize": 51836776, "license": "other", "mime_type": "application/pdf", "url": "/6797/1/Nunez_me_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Oxidation of DNA by Long-Range Charge Transport", "author": [ { "family_name": "Nu\u00f1ez", "given_name": "Megan Elizabeth", "clpid": "Nu\u00f1ez-Megan-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ever since the double helical structure of DNA was elucidated, it has been proposed that charge might move through the stacked base pairs of the double helix because of the electronic coupling of the \u03c0 orbitals of the nucleotide bases with neighboring bases. Here it is demonstrated that electronic \"holes\" generated\r\nby a one-electron oxidation of DNA can result in permanent lesions on guanine bases up to 200 \u00c5 away from the intercalating oxidant as a result of such charge\r\nmigration. Both rhodium and ruthenium complexes, covalently tethered to the 5' end of a double-stranded oligonucleotide and intercalated into the base stack, can with photoactivation promote oxidation of guanines in 5'-GG-3' sites over this distance. Since charges can move efficiently through the DNA oligonucleotides, it was important to characterize this reaction in more detail, and to extend observations of charge transport through DNA to larger and more complicated DNA assemblies that more closely mimic its structure in vivo.
\r\n\r\nLong-range oxidative damage to guanine doublets in DNA is shown to compete for oxidation with other reactions, such as the repair of thymine dimers. When both thymine dimer lesions and guanine doublets are present, both can be\r\noxidized by a photoexcited rhodium complex, although each in lower yield than in the absence of the other. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Therefore electronic \"holes\" generated on genomic DNA might not of necessity cause DNA damage, but could also be funneled onto proteins or other oxidizible sites.
\r\n\r\nUsing a variety of intercalating photooxidants targeted to a specific site on a restriction fragment by an appended triplex-forming oligonucleotide, the upper distance limits and sequence effects on long-range charge transfer through DNA were examined. Charge migration occurs in both directions from the intercalator and on both DNA strands of the target, but the oxidation is significantly more\r\nefficient to the 3' side of the triplex, over 25-38 base pairs. When intercalators were tethered directly to the 5' terminus of the triplex-forming strand as opposed\r\nto the center, significant amounts of oxidative damage was generated only in the immediate vicinity of the intercalation site, suggesting that the base stack is\r\ndistorted at the 5' end of the triplex region in the duplex/triplex junction. Targeting of photooxidative damage by triplex formation extends previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed.
\r\n\r\nWithin eukaryotic cells most DNA is packaged as nucleosome core particles, made up of ~146 base pairs of DNA wrapped around a core of histone proteins. Photoexcited rhodium complexes were also used to explore charge\r\ntransport through DNA within these structures. Although histone proteins inhibit intercalation of a noncovalent rhodium complex, they do not prevent oxidation of\r\n5'-GG-3' sites, the signature of oxidative charge transport through DNA. Furthermore, some of these sites are not directly accessible to a solution-bound oxidant due to his tones in the major groove, and thus they must be oxidized from a distance. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage from solution-borne molecules, it does not protect the DNA from charge transfer damage through the base pair stack. In\r\nsupport of this assertion, guanine bases within nucleosomal DNA were oxidized at a distance of over 23 base pairs from a covalently-tethered rhodium intercalator.
\r\n\r\nThe environment within the cell nucleus contains a variety of other proteins and small molecules that could potentially influence the migration of charge through DNA. Using the rhodium photochemistry, the oxidation of\r\nguanine by photoexcited rhodium complexes inside of nuclei from cultured human cells was examined and compared with the oxidative damage on bare genomic DNA. Oxidation occurs preferentially at the 5'-guanine of 5'-GG-3' sites, indicative of base damage by DNA-mediated charge transport chemistry. Moreover, oxidative damage occurs at protein-bound sites which are inaccessible to rhodium. Thus, on transcriptionally active DNA within the cell nucleus, DNA-mediated charge transport acts to induce base damage from a distance. Direct interaction of an oxidant is not necessary to generate a base lesion at a specific\r\nsite within the nucleus.
\r\n\r\nAll of these observations indicate that charges can migrate along DNA within the cell. These observations require a reconsideration of cellular mechanisms for DNA damage and repair, and present new avenues for exploration in the design of DNA-based drugs and therapies.
", "doi": "10.7907/ZQ5T-9Z15", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:330", "collection": "thesis", "collection_id": "330", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01252002-100801", "primary_object_url": { "basename": "bolon_thesis.pdf", "content": "final", "filesize": 4053155, "license": "other", "mime_type": "application/pdf", "url": "/330/1/bolon_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Computational Enzyme Design", "author": [ { "family_name": "Bolon", "given_name": "Daniel N.", "orcid": "0000-0001-5857-6676", "clpid": "Bolon-Daniel-N" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The long-term objective of computational enzyme design is the ability to generate efficient protein catalysts for any chemical reaction. This thesis develops and experimentally validates a general computational approach for the design of enzymes with novel function.
\r\n \r\nIn order to include catalytic mechanism in protein design, a high-energy state (HES) rotamer (side chain representation) was constructed. In this rotamer, substrate atoms are in a HES. In addition, at least one amino acid side chain is positioned to interact favorably with substrate atoms in their HES and facilitate the reaction. Including an amino acid side chain in the HES rotamer automatically positions substrate relative to a protein scaffold and allows protein design algorithms to search for sequences capable of interacting favorably with the substrate. Because chemical similarity exists between the transition state and the high-energy state, optimizing the protein sequence to interact favorably with the HES rotamer should lead to transition state stabilization. In addition, the HES rotamer model focuses the subsequent computational active site design on a relevant phase space where an amino acid is capable of interacting in a catalytically active geometry with substrate.
\r\n\r\nUsing a HES rotamer model of the histidine mediated nucleophilic hydrolysis of p-nitrophenyl acetate, the catalytically inert 108 residue E. coli thioredoxin as a scaffold, and the ORBIT protein design software to compute sequences, an active site scan identified two promising active site designs. Experimentally, both candidate ?protozymes? demonstrated catalytic activity significantly above background. In addition, the rate enhancement of one of these ?protozymes? was the same order of magnitude as the first catalytic antibodies.
\r\n\r\nBecause polar groups are frequently buried at enzyme-substrate interfaces, improved modeling of buried polar interactions may benefit enzyme design. By studying native protein structures, rules have been developed within the scope of protein design that require core polar residues to largely satisfy their hydrogen bonding potential. Using this polar strategy to design the core of thioredoxin resulted in a protein that was thermodynamically stabilized relative to both the wt protein and a protein designed without core polar residues.
\r\n\r\nThe enzyme design procedures presented here may serve as a platform to develop more detailed methods. It is hoped that the development and experimental testing of more detailed methods will continue to improve our understanding of enzyme mechanism and lead to the long-term goal of designing highly efficient enzymes.
", "doi": "10.7907/W7F3-DS14", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6813", "collection": "thesis", "collection_id": "6813", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02072012-093202232", "primary_object_url": { "basename": "Yeh_ap_2002.pdf", "content": "final", "filesize": 36332899, "license": "other", "mime_type": "application/pdf", "url": "/6813/1/Yeh_ap_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Crystallographic Studies of Iron Proteins", "author": [ { "family_name": "Yeh", "given_name": "Andrew Peter", "orcid": "0000-0003-4610-4940", "clpid": "Yeh-Andrew-Peter" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The crystal structures of a number of iron proteins from various microbes have been determined in order to better understand the structure-function relationship of these proteins. Several of these iron proteins are as follows:
\r\n\r\nSuperoxide Reductase (SOR) from the hyperthermophile Pyrococcus furiosus. SOR is a non-heme mono-iron protein that functions in anaerobic microbes (e.g., Pyrococcus furiosus) as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide. Crystal structures of SOR in both its oxidized and reduced states have been determined and suggest a possible mechanism by which superoxide accessibility may be regulated.
\r\n\r\n\r\n[2Fe-2S] Ferredoxin 4 (Fd4) from the hyperthermophile Aquifex aeolicus. The crystal structure of this [2Fe-2S] ferredoxin has been determined and reveals a thioredoxin-like fold that is novel among iron-sulfur proteins. Protein sequence alignments show that this fold is present as components of more complex anaerobic and aerobic electron transfer systems (e.g., complex I of aerobic respiratory chains). The crystal structures of two variants of this protein in which one of the [2Fe-2S] cysteine ligands was substituted with a serine have also been determined. The structures of these variants provide metric details of unprecedented accuracy for serine-ligated iron-sulfur clusters in proteins.
\r\n\r\n\r\nThe Photosynthetic Reaction Center (RC) from the photosynthetic purple bacterium Rhodobacter sphaeroides. The plimary process of bacterial photosynthesis, which is light-induced trans-membrane charge separation, occurs in the reaction center (RC), an integral membrane protein-pigment complex. We have obtained the crystal structures of the RC bound to the inhibitor stigmatellin in the presence and absence of light to determine any structural change(s) that may be associated with one of its light-induced charge-separated (D^+QA^-) states. In addition, we have determined the crystal structure of the RC complexed with its physiological electron donor, the soluble monoheme protein cytochrome C_2.
", "doi": "10.7907/25dt-ct33", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6779", "collection": "thesis", "collection_id": "6779", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01252012-151300693", "primary_object_url": { "basename": "Belitsky_jm_2002.pdf", "content": "final", "filesize": 49802669, "license": "other", "mime_type": "application/pdf", "url": "/6779/1/Belitsky_jm_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA Binding Polyamides in Biological Systems", "author": [ { "family_name": "Belitsky", "given_name": "Jason Matthew", "clpid": "Belitsky-Jason-Matthew" } ], "thesis_advisor": [ { "family_name": "Janda", "given_name": "Kenneth C.", "clpid": "Janda-K-C" } ], "thesis_committee": [ { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Janda", "given_name": "Kenneth C.", "clpid": "Janda-K-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind selectively to a DNA sequence in the human genome are potentially useful tools for molecular biology and human medicine. Polyamides containing\r\nN-methylimidazole (Im) and N-methylpyrrole (Py) are small molecules that bind DNA according to a set of \"pairing rules\" with affinities and specificities that rival natural transcription factors. By directly competing with a given transcription factor or other DNA binding protein for its binding site, polyamide can cause inhibition of diverse biological processes, such as retroviral integration and gene transcription. Polyamides are presented which inhibit the in vitro integration activities for two retroviruses, M-MuLV and HIV-l. Polyamides are described that inhibit TBP binding to the HER2 promoter, a gene implicated in human breast cancer. Failure to achieve inhibition of HER2 transcription in cell culture led to the surprising discovery that polyamide-fluorescent dye conjugates are cell permeable, but that nuclear localization does not occur in many cell lines. Efforts toward modified polyamides with enhanced nuclear localization properties are presented. In order to extend the number of sequences amenable to high affinity recognition, alteration of the C-terminal polyamide tail has been investigated. The development of conditions for polyamide solid-phase synthesis on a new resin, which allows for the generation of \"truncated tail\" polyamides, is presented. During the original route to one of these compounds, an\r\nunexpected reaction was uncovered that leads to entirely different C-terminal tails.", "doi": "10.7907/WNV6-DH45", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:8161", "collection": "thesis", "collection_id": "8161", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03242014-152154971", "primary_object_url": { "basename": "Zubris_dl_2001.pdf", "content": "final", "filesize": 51236605, "license": "other", "mime_type": "application/pdf", "url": "/8161/1/Zubris_dl_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigations of the Origin of Stereocontrol in Syndiospecific Ziegler-Natta Polymerizations", "author": [ { "family_name": "Zubris", "given_name": "Deanna Lynn", "orcid": "0000-0003-1003-9630", "clpid": "Zubris-Deanna-Lynn" } ], "thesis_advisor": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In order to expand our understanding of the mechanism of stereocontrol\r\nin syndiospecific \u03b1-olefin polymerization, a family of Cs-symmetric, ansa-group 3\r\nmetallocenes was targeted as polymerization catalysts. The syntheses of new\r\nansa-yttrocene and scandocene derivatives that employ the doubly [SiMe2]-\r\nbridged ligand array (1,2-SiMe2)2{C5H-3,5-(CHMe2)2} (where R = t-\r\nbutyl, tBuThp; where R = i-propyl, iPrThp) are described. The structures of\r\ntBuThpY(\u00b5-Cl)2K(THF)2, tBuThpSc(\u00b5-Cl)2K(Et2O)2, tBuThpYCH(SiMe3)2, Y2{\u00b52-(tBuThp)2}(\u00b52-H)2, and tBuThpSc(\u00b5-CH3)2 have been examined by\r\nsingle crystal X-ray diffraction methods. Ansa-yttrocenes and scandocenes that\r\nincorporate the singly [CPh2]-bridged ligand array (CPh2)(C5H4)(C13H8)(where\r\nC5H4 = Cp, cyclopentadienyl; where C13H8 = Flu, fluourenyl) have also been\r\nprepared. Select meallocene alkyl complexes are active single component\r\ncatalysts for homopolymerization of propylene and 1-pentene. The scandocene\r\ntetramethylaluminate complexes generate polymers with the highes molecular\r\nweights of the series. Under all conditions examined atactic polymer\r\nmicrostructures are observed, suggesting a chain-end mechanism for\r\nstereocontrol.
\r\n\r\nA series of ansa-tantalocenes have been prepared as models for Ziegler-Natta\r\npolymerization catalysts. A singly bridged ansa-tantalocene trimethyl\r\ncomplex, Me2Si(\u03b75-C5H4)2TaMe3, has been prepared and used for the synthesis\r\nof a tantalocene ethylene-methyl complex. Addition of propylene to this\r\nethylene-methyl adduct results in olefin exchange to give a mixture of endo and\r\nexo propylene isomers. Doubly-silylene bridged ansa-tantalocene complexes\r\nhave been prepared with the tBuThp ligand; a tantalocene trimethyl complex and\r\na tantalocene methylidene-methyl complex have been synthesized and\r\ncharacterized by X-ray diffraction. Thermolysis of the methylidene-methyl\r\ncomplex affords the corresponding ethylene-hydride complex. Addition of\r\neither propylene or styrene to this ethylene-hydride compound results in olefin\r\nexchange. In both cases, only one product isomer is observed. Studies of olefin\r\nexchange with ansa-tantalocene olefin-hydride and olefin-methyl complexes have\r\nprovided information about the important steric influences for olefin\r\ncoordination in Ziegler-Natta polymerization.
\r\n\r\n", "doi": "10.7907/j7gk-0s22", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:5372", "collection": "thesis", "collection_id": "5372", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11122009-154318044", "primary_object_url": { "basename": "Debe_da_2001.pdf", "content": "final", "filesize": 6323465, "license": "other", "mime_type": "application/pdf", "url": "/5372/1/Debe_da_2001.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Shaving Levinthal with Occam's Razor: Understanding the Rate Limiting Step in Protein Folding", "author": [ { "family_name": "Debe", "given_name": "Derek Anthony", "clpid": "Debe-Derek-Anthony" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "How do proteins fold? This thesis addresses this simple yet important question by developing a first principles theoretical framework that accurately describes the experimentally observed protein folding rate data. The success of the new theory suggests that single domain proteins fold according two a two- state mechanism consisting of
\r\n(i)\ta random, diffusive search for the native topology, followed by
\r\n(ii)\tnon-random, local conformation changes within the native topology to find the unique native state.
In chapter 1, a popular analogy between protein folding and the game of golf is used to qualitatively illustrate the most important aspects of the new theory. In chapter 2, mean-field computational methods are developed that allow the time involved in the rate limiting diffusive search for the native state to be calculated. Chapters 3 and 4 remove the mean-field restriction from the methods of chapter 2, allowing the folding rate for an arbitrary two-state folding protein to be calculated. Chapter 5 then explores how real proteins deviate from this ideal model by examining the roles that non-random mechanisms such as helix, hydrophobic core, and 13-turn formation play in the early folding process. Finally, chapter 6 develops an empirical model that also capably predicts protein folding rates, adding further support to the proposed folding mechanism.
", "doi": "10.7907/p4yc-y834", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8116", "collection": "thesis", "collection_id": "8116", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03072014-143524462", "type": "thesis", "title": "Protein-Protein Recognition: The Neonatal Fc Receptor and Immunoglobulin G", "author": [ { "family_name": "Martin", "given_name": "Warham Lance", "clpid": "Martin-Warham-Lance" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The neonatal Fc receptor (FcRn) binds the Fc portion of immunoglobulin G (IgG)\r\nat the acidic pH of endosomes or the gut and releases IgG at the alkaline pH of blood.\r\nFcRn is responsible for the maternofetal transfer of IgG and for rescuing endocytosed\r\nIgG from a default degradative pathway. We investigated how FcRn interacts with IgG\r\nby constructing a heterodimeric form of the Fc (hdFc) that contains one FcRn binding\r\nsite. This molecule was used to characterize the interaction between one FcRn molecule\r\nand one Fc and to determine under what conditions FcRn forms a dimer. The hdFc binds\r\none FcRn molecule at pH 6.0 with a Kd of 80 nM. In solution and with FcRn anchored to\r\nsolid supports, the heterodimeric Fc does not induce a dimer of FcRn molecules. FcRnhdFc\r\ncomplex crystals were obtained and the complex structure was solved to 2.8 \u00c5\r\nresolution. Analysis of this structure refined the understanding of the mechanism of the\r\npH-dependent binding, shed light on the role played by carbohydrates in the Fc binding,\r\nand provided insights on how to design therapeutic IgG antibodies with longer serum\r\nhalf-lives. The FcRn-hdFc complex in the crystal did not contain the FcRn dimer. To\r\ncharacterize the tendency of FcRn to form a dimer in a membrane we analyzed the\r\ntendency of the hdFc to induce cross-phosphorylation of FcRn-tyrosine kinase chimeras.\r\nWe also constructed FcRn-cyan and FcRn-yellow fluorescent proteins and have analyzed\r\nthe tendency of these molecules to exhibit fluorescence resonance energy transfer. As of\r\nnow, neither of these analyses have lead to conclusive results. In the process of acquiring\r\nthe context to appreciate the structure of the FcRn-hdFc interface, we developed a study\r\nof 171 other nonobligate protein-protein interfaces that includes an original principal\r\ncomponent analysis of the quantifiable aspects of these interfaces.", "doi": "10.7907/9ek6-6833", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8119", "collection": "thesis", "collection_id": "8119", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03102014-140846235", "primary_object_url": { "basename": "Liu 2001.pdf", "content": "final", "filesize": 89772038, "license": "other", "mime_type": "application/pdf", "url": "/8119/1/Liu 2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Molecular Mechanism of Sulfated Carbohydrate Recognition: Structural and Biochemical Studies of the Cysteine-Rich Domain of Mannose Receptor", "author": [ { "family_name": "Liu", "given_name": "Yang", "clpid": "Liu-Yang-Biology" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Mannose receptor (MR) is widely expressed on macrophages, immature dendritic\r\ncells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane\r\nreceptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich\r\ndomain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type\r\nlectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct\r\ncarbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the\r\ntandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc).\r\nThe dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated\r\npolysaccharide chains, and thereby facilitating the involvement of MR in\r\nimmunological and physiological processes. The immunological functions of MR include\r\nantigen capturing (through binding non-sulfated carbohydrates) and antigen targeting\r\n(through binding sulfated carbohydrates), and the physiological roles include rapid clearance\r\nof circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with\r\nsulfated and non-sulfated carbohydrates.
\r\n\r\nWe have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0\r\n\u00c5) and Cys-MR complexed with different ligands, including Hepes (1.7 \u00c5), 4SO_4-N-Acetylgalactosamine\r\n(4SO_4-GalNAc; 2.2 \u00c5), 3SO_4-Lewis^x (2.2 \u00c5), 3S04-Lewis^a (1.9 \u00c5),\r\nand 6SO_4-GalNAc (2.5 \u00c5). The overall structure of Cys-MR consists of 12 anti-parallel \u03b2-strands\r\narranged in three lobes with approximate three fold internal symmetry. The structure\r\ncontains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The\r\nligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate\r\ngroup of ligand is buried. Our results show that optimal binding is achieved by a\r\ncarbohydrate ligand with a sulfate group that anchors the ligand by forming numerous\r\nhydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR.\r\nUsing a fluorescence-based assay, we characterized the binding affinities between CysMR\r\nand its ligands, and rationalized the derived affinities based upon the crystal structures.\r\nThese studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and\r\nfacilitate our understanding of the role of Cys-MR in MR recognition of its ligands.
\r\n", "doi": "10.7907/fj0v-hx76", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8137", "collection": "thesis", "collection_id": "8137", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03142014-154815404", "primary_object_url": { "basename": "Chang 2001.pdf", "content": "final", "filesize": 16564703, "license": "other", "mime_type": "application/pdf", "url": "/8137/1/Chang 2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Cleavage of DNA by Polamide-Seco-CB1 Conjugates", "author": [ { "family_name": "Chang", "given_name": "Aileen Yulin", "clpid": "Chang-Aileen-Yulin" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind to any predetermined DNA sequence in the human genome are potentially useful tools for molecular biology and human medicine. Polyamides containing N-methylimidazole (Im) N-methylpyrrole (Py) are cell permeable small molecules that bind DNA according to a set of \"pairing rules\" with affinities and specificities similar to many naturally occurring DNA binding proteins. Py-Im polyamides offer a general approach to the chemical regulation of gene expression. We demonstrate here that polyamide containing a DNA alkylating moiety seco-CBI can specifically direct sequence specific DNA alkylation. We can also control the strand of DNA that is alkylated, depending on the enantiomer of seco-CBI used and the orientation of the polyamide relative to the alkylation site (Chapter 2). This class of molecules has been applied to a gene repair system in collaboration with the Baltimore group at Caltech (Chapter 3). Also reported are additional seco-CBI polyamide conjugates synthesized to study other systems (HIV-1 and COX-2) (Appendix 1).\r\n", "doi": "10.7907/FY0F-DW57", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8151", "collection": "thesis", "collection_id": "8151", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03202014-093625161", "primary_object_url": { "basename": "Krider_es_2001.pdf", "content": "final", "filesize": 51581938, "license": "other", "mime_type": "application/pdf", "url": "/8151/1/Krider_es_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Spectroscopy of Ruthenium-Modified Nucleic Acids", "author": [ { "family_name": "Krider", "given_name": "Elizabeth Stratford", "clpid": "Krider-Elizabeth-Stratford" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Meade", "given_name": "Thomas J.", "clpid": "Meade-T-J" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Redox-active probes are designed and prepared for use in DNA-mediated electron\r\ntransfer studies. These probes consist of ruthenium(II) complexes bound to nucleosides\r\nthat possess metal-binding ligands. Low- and high-potential oxidants are synthesized\r\nfrom these modified nucleosides and display reversible one-electron electrochemical\r\nbehavior. The ruthenium-modified nucleosides exhibit distinct charge-transfer transitions\r\nin the visible region that resemble those of appropriate model complexes. Resonance\r\nRaman and time-resolved emission spectroscopy are used to characterize the nature of\r\nthese transitions.
\r\n\r\nThe site-specific incorporation of these redox-active probes into oligonucleotides\r\nis explored using post-synthetic modification and solid-phase synthetic methods. The\r\npreparation of the metal-binding nucleosides, their incorporation into oligonucleotides,\r\nand characterization of the resulting oligonucleotides is described. Because the insertion\r\nof these probes into modified oligonucleotides using post-synthetic modification is\r\nunsuccessful, solid-phase synthetic methods are explored. These efforts lead to the first\r\nreport of 3'-metallated oligonucleotides prepared completely by automated solid-phase\r\nsynthesis. Preliminary efforts to prepare a bis-metallated oligonucleotide by automated\r\nsynthesis are described.
\r\n\r\nThe electrochemical, absorption, and emissive features of the ruthenium-modified\r\noligonucleotides are unchanged from those of the precursor metallonucleoside. The\r\nabsence of any change in these properties upon incorporation into oligonucleotides and\r\nsubsequent hybridization suggests that the incorporated ruthenium(II) complex is a\r\nvaluable probe for DNA-mediated electron transfer studies.
", "doi": "10.7907/s0za-ec86", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8154", "collection": "thesis", "collection_id": "8154", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03202014-144611863", "primary_object_url": { "basename": "Machczynski_mc_2001.pdf", "content": "final", "filesize": 16647250, "license": "other", "mime_type": "application/pdf", "url": "/8154/1/Machczynski_mc_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Physical Characterization of the Rack Effect and Hydrogen Bond Networks in Blue Copper Proteins", "author": [ { "family_name": "Machczynski", "given_name": "Michael Christopher", "clpid": "Machczynski-Michael-Christopher" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A summary of previous research is presented that indicates that the purpose of a\r\nblue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a\r\ncopper(II) geometry around the copper(I) ion in the metal site. In several blue copper\r\nproteins, the C-terminal histidine ligand becomes protonated and detaches from the\r\ncopper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were\r\nmade to alter the hydrogen bond network and quantify the rack effect by pKa shifts.
\r\n\r\nThe pKa's of mutant amicyanins have been measured by pH-dependent\r\nelectrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the\r\nreduced copper to adopt a relaxed conformation: the ability to relax drives the reduction\r\npotentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F)\r\nmV vs. NHE. The measured pKa's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The\r\nadditional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the\r\nblue copper absorption and an increase in the parallel hyperfine splitting in the EPR\r\nspectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal\r\nimidazole.
\r\n\r\nMelting curves give a measure of the thermal stability of the protein. A\r\nthermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons\r\nwith the electrochemistry and apoamicyanin suggest that the intermediate involves the\r\nregion of the protein near the metal site. This region is destabilized in the P94F mutant;\r\ncoupled with the evidence that the imidazole is stabilized under the same conditions\r\nconfirms an original concept of the rack effect: a high energy configuration is stabilized\r\nat a cost to the rest of the protein.
", "doi": "10.7907/q0yk-sj10", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8156", "collection": "thesis", "collection_id": "8156", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212014-084556805", "primary_object_url": { "basename": "HUNG.pdf", "content": "final", "filesize": 41513990, "license": "other", "mime_type": "application/pdf", "url": "/8156/1/HUNG.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Succinate: Ubiquinone Oxidoreductase from Paracoccus denitrificans and Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath): Experimental and Theoretical EPR Studies of the Metal Cofactors", "author": [ { "family_name": "Hung", "given_name": "Shao-Ching", "clpid": "Hung-Shao-Ching" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis summarizes the application of conventional and modern electron paramagnetic resonance (EPR) techniques to establish proximity relationships between paramagnetic metal centers in metalloproteins and between metal centers and magnetic ligand nuclei in two important and timely membrane proteins: succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans and particulate methane\r\nmonooxygenase (pMMO) from Methylococcus capsulatus. Such proximity relationships are thought to be critical to the biological function and the associated biochemistry mediated by the metal centers in these proteins. A mechanistic understanding of biological function relies heavily on structure-function relationships and the knowledge of how molecular structure and electronic properties of the metal centers influence the reactivity in metalloenzymes. EPR spectroscopy has proven to be one of the most powerful techniques towards obtaining information about interactions between metal centers as well as defining ligand structures. SQR is an electron transport enzyme wherein the substrates, organic and metallic cofactors are held relatively far apart. Here, the proximity relationships of the metallic cofactors were studied through their weak spin-spin interactions by means of EPR power saturation and electron spin-lattice (T_1)\r\nmeasurements, when the enzyme was poised at designated reduction levels. Analysis of the electron T_1 measurements for the S-3 center when the b-heme is paramagnetic led to\r\na detailed analysis of the dipolar interactions and distance determination between two interacting metal centers. Studies of ligand environment of the metal centers by electron spin echo envelope modulation (ESEEM) spectroscopy resulted in the identication of peptide nitrogens as coupled nuclei in the environment of the S-1 and S-3 centers.
\r\n\r\nFinally, an EPR model was developed to describe the ferromagnetically coupled trinuclear copper clusters in pMMO when the enzyme is oxidized. The Cu(II) ions in these clusters appear to be strongly exchange coupled, and the EPR is consistent with equilateral triangular arrangements of type 2 copper ions. These results offer the first glimpse of the magneto-structural correlations for a trinuclear copper cluster of this type, which, until the work on pMMO, has had no precedent in the metalloprotein literature. Such trinuclear copper clusters are even rare in synthetic models.
\r\n", "doi": "10.7907/ze04-mx93", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8156", "collection": "thesis", "collection_id": "8156", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212014-084556805", "primary_object_url": { "basename": "HUNG.pdf", "content": "final", "filesize": 41513990, "license": "other", "mime_type": "application/pdf", "url": "/8156/1/HUNG.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Succinate: Ubiquinone Oxidoreductase from Paracoccus denitrificans and Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath): Experimental and Theoretical EPR Studies of the Metal Cofactors", "author": [ { "family_name": "Hung", "given_name": "Shao-Ching", "clpid": "Hung-Shao-Ching" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis summarizes the application of conventional and modern electron paramagnetic resonance (EPR) techniques to establish proximity relationships between paramagnetic metal centers in metalloproteins and between metal centers and magnetic ligand nuclei in two important and timely membrane proteins: succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans and particulate methane\r\nmonooxygenase (pMMO) from Methylococcus capsulatus. Such proximity relationships are thought to be critical to the biological function and the associated biochemistry mediated by the metal centers in these proteins. A mechanistic understanding of biological function relies heavily on structure-function relationships and the knowledge of how molecular structure and electronic properties of the metal centers influence the reactivity in metalloenzymes. EPR spectroscopy has proven to be one of the most powerful techniques towards obtaining information about interactions between metal centers as well as defining ligand structures. SQR is an electron transport enzyme wherein the substrates, organic and metallic cofactors are held relatively far apart. Here, the proximity relationships of the metallic cofactors were studied through their weak spin-spin interactions by means of EPR power saturation and electron spin-lattice (T_1)\r\nmeasurements, when the enzyme was poised at designated reduction levels. Analysis of the electron T_1 measurements for the S-3 center when the b-heme is paramagnetic led to\r\na detailed analysis of the dipolar interactions and distance determination between two interacting metal centers. Studies of ligand environment of the metal centers by electron spin echo envelope modulation (ESEEM) spectroscopy resulted in the identication of peptide nitrogens as coupled nuclei in the environment of the S-1 and S-3 centers.
\r\n\r\nFinally, an EPR model was developed to describe the ferromagnetically coupled trinuclear copper clusters in pMMO when the enzyme is oxidized. The Cu(II) ions in these clusters appear to be strongly exchange coupled, and the EPR is consistent with equilateral triangular arrangements of type 2 copper ions. These results offer the first glimpse of the magneto-structural correlations for a trinuclear copper cluster of this type, which, until the work on pMMO, has had no precedent in the metalloprotein literature. Such trinuclear copper clusters are even rare in synthetic models.
\r\n", "doi": "10.7907/ze04-mx93", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8157", "collection": "thesis", "collection_id": "8157", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212014-095851500", "primary_object_url": { "basename": "odom 2001.pdf", "content": "final", "filesize": 39129217, "license": "other", "mime_type": "application/pdf", "url": "/8157/1/odom 2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Application of Metallointercalators in Recognition of and Charge Transport in Nucleic Acids", "author": [ { "family_name": "Odom", "given_name": "Duncan T.", "orcid": "0000-0001-6201-5599", "clpid": "Odom-Duncan-T" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety\r\nbind to DNA through the major groove. These metallointercalators can recognize DNA\r\nsites and perform reactions on DNA as a substrate. The site-specific metallointercalator\r\n\u039b-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription\r\nfactor, yAP-1, from an oligonucleotide that contains a common binding site. The\r\ndemonstration that metal complexes can prevent transcription factor binding to DNA site-specifically\r\nis an important step in using metallointercalators as therapeutics.
\r\n\r\nThe distinctive photochemistry of metallointercalators can also be applied to\r\npromote long range charge transport in DNA. Experiments using duplexes with regions\r\n4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying\r\norder showed that radical migration is more dependent on the sequence of bases, and\r\nless dependent on the distance between the guanine doublets. This result suggests that\r\nmechanistic proposals of long range charge transport must involve all the bases.
\r\n\r\nRNA/DNA hybrids show charge migration to guanines from a remote site, thus\r\ndemonstrating that nucleic acid stacking other than B-form can serve as a radical bridge.\r\nDouble crossover DNA assemblies also provide a medium for charge transport at\r\ndistances up to 100 \u00c5 from the site of radical introduction by a tethered metal complex.\r\nThis radical migration was found to be robust to mismatches, and limited to individual,\r\nelectronically distinct base stacks. In single DNA crossover assemblies, which have\r\nconsiderably greater flexibility, charge migration proceeds to both base stacks due to\r\nconformational isomers not present in the rigid and tightly annealed double crossovers.
\r\n\r\nFinally, a rapid, efficient, gel-based technique was developed to investigate\r\nthymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated\r\nvia the bases of a thymine-thymine interface; reversal of this ligation is easily visualized\r\nby gel electrophoresis. This assay was used to show that the repair of thymine dimers\r\nfrom a distance through DNA charge transport can be accomplished with different\r\nphotooxidants.
\r\n\r\nThus, nucleic acids that support long range charge transport have been shown to\r\ninclude A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a\r\nmethod for thymine dimer repair detection using charge transport was developed.\r\nThese observations underscore and extend the remarkable finding that DNA can serve a\r\nmedium for charge transport via the heteroaromatic base stack.
\r\n\r\n", "doi": "10.7907/yz1f-w961", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:11371", "collection": "thesis", "collection_id": "11371", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-135136732", "type": "thesis", "title": "Reactions of Heme Proteins to Solutions and Crystals", "author": [ { "family_name": "Tezcan", "given_name": "Faik Akif", "orcid": "0000-0002-4733-6500", "clpid": "Tezcan-Faik-Akif" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "orcid": "0000-0002-4453-9716", "clpid": "Winkler-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "To assess the effects of heme solvation and iron ligation on reduction potentials in c-type cytochromes, we have examined the redox and ligand-binding properties of microperoxidase-8 (MP8). Methionine-, histidine- and amine-coordination to MP8 were found to account for 130, -40 and -10-mV shifts in the Fe(III/II)-potential, respectively. Our finding that reduction potentials increase with decreasing heme-surface exposure suggests that the protein matrix can further tune the reduction potential by 500 mV through water exclusion from the heme pocket.
\r\n\r\nThe 410-mV upshift in the cytochrome c (cyt c) potential as the heme cofactor is moved from a highly-solvated environment to the protein interior signals a 10-kcal/mol greater stability of the reduced form. Consequently, there exists a range of denaturant concentrations where Fe(II)-cyt c is folded and Fe(III)-cyt c is unfolded. Electron injection into the oxidized protein in this range triggers the folding reaction. Using NADH as a redox photosensitizer, cyt c folding can be initiated within 100 \u00b5s. Our results suggest that the folding of cyt c is rate-limited by ligand-substitution events on the iron center.
\r\n\r\nDue to an increased barrier to ligand substitution, folding of Co(III)-substituted cyt cis 5 orders of magnitude slower than Fe-cyt c. The slow folding kinetics of Co(III)-cyt c have allowed the convenient study of protein dynamics with a variety of spectroscopic techniques, revealing previously unresolved folding pathways involving Lys- and His-misligated populations of the unfolded molecule and extremely long-lived folding intermediates.
\r\n\r\nFactors that control electron flow between proteins are not well understood, owing to uncertainties in the relative orientations and structures of the reactants during the short time that tunneling occurs. To circumvent this ambiguity, we have measured the kinetics of electron transfer (ET) between native and Zn-substituted tuna cyt c molecules in crystals of known structure. ET rates (320 s-1 for *Zn-cyt c \u2192 Fe(III)-cyt c; 2000 s-1 for Fe(II)-cyt c \u2192 Zn-cyt c+) over a Zn-Fe distance of 24.1 \u00c5 closely match those for intraprotein ET over similar donor-acceptor separations. Our results indicate that van der Waals interactions and water mediated H-bonds provide effective electronic coupling across a protein-protein interface.
", "doi": "10.7907/rrqd-9v38", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:4932", "collection": "thesis", "collection_id": "4932", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12102007-114331", "primary_object_url": { "basename": "Miller_ma_2001.PDF", "content": "final", "filesize": 67396101, "license": "other", "mime_type": "application/pdf", "url": "/4932/1/Miller_ma_2001.PDF", "version": "v3.0.0" }, "type": "thesis", "title": "Laser Synchronized Optical Nuclear Magnetic Resonance via Larmor Beat Detection : Imaging Electronic Wavefunctions in Gallium Arsenide Device Structures", "author": [ { "family_name": "Miller", "given_name": "Michael Andrew", "clpid": "Miller-Michael-Andrew" } ], "thesis_advisor": [ { "family_name": "Weitekamp", "given_name": "Daniel P.", "orcid": "0000-0003-0079-8000", "clpid": "Weitekamp-D-P" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Weitekamp", "given_name": "Daniel P.", "orcid": "0000-0003-0079-8000", "clpid": "Weitekamp-D-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "We have accomplished Optical Nuclear Magnetic Resonance (ONMR) experiments in an Al0.36Ga0.64As/GaAs heterojunction sample at ~2K with rf-optical pulse synchronization. The hyperfine coupling of the electron spin to the nuclear spins enable this spectroscopy in several ways, which are discussed herein. Moreover, the interactions experienced by nuclear spins in III-V semiconductors, in general, and the phenomena encountered when they are in the vicinity of a shallow donor or pseudo-donor, specifically, are developed. Furthermore, the most accurate calculation of spin diffusion in a spin-three-halves system to date is developed and presented using a methodology can be readily applied to any spin-larger-than-one-half system to a yield a set of coupled differential equations for a set of orthogonal polarizations. The behavior of these equations under a number of physical situations is also investigated.
\r\n\r\nWe have captured the first ever radially resolved Knight shift images from the nuclei near a point defect in GaAs using laser synchronized ONMR. A deconvolution of these images into their constituent physical interactions has been approximately carried out using the theoretical advances developed and presented in this thesis, yielding the shape and size of the electronic orbital in which the electron is trapped, the occupancy of that electronic orbital, and the quadrupolar interactions in the vicinity of the defect, including the charge state of the defect.
\r\n\r\nComputational approaches include both full, real-time analyses of every one of the hundreds of thousands of nuclei surrounding a defect in GaAs, modeling the time domain evolution for each individual nucleus including its Knight shift, quadrupolar interactions (both secular and nonsecular), individual optical polarization conditions, optical detection weighting, and rigorously exact rf effects, and analyses of a variety of continuous medium approximations. The only computations that fit the experimental spectra are those that calculate spin diffusion along a radial line of spins, and use this approximation to the radial profile of nuclear polarization in a continuous medium approximation. The successful interface of this spin diffusion calculation and the single nucleus calculations, leveraging their individual strengths, is clearly a desirable route to further increase computational accuracy.
", "doi": "10.7907/ARCE-F837", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:5386", "collection": "thesis", "collection_id": "5386", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11192009-084608026", "primary_object_url": { "basename": "Carlson_mj_2000.pdf", "content": "final", "filesize": 9021815, "license": "other", "mime_type": "application/pdf", "url": "/5386/1/Carlson_mj_2000.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "BUFF: A Biological Universal Forcefield Derived from Quantum Mechanics", "author": [ { "family_name": "Carlson", "given_name": "Matt Jeffrey", "clpid": "Carlson-Matt-Jeffrey" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Molecular mechanical simulations of biomolecules require an accurate potential energy function (forcefield) in order to produce meaningful results. Most current forcefields are highly parameterized in order to correctly reproduce high level theory and experiment. Increasingly, new biomolecules are designed and studied that have atypical configurations such as metal centers and nonstandard amino acids. To avoid a lengthy process to develop new parameters for each new system encountered, a generic forcefield is desired. A hierarchical approach is undertaken herein to achieve this flexibility and accuracy.\r\n\r\nBuilding upon the rule based generic forcefields UFF and Dreiding, a new biological universal forcefield, BUFF, is presented for the simulation of proteins and other biological molecules. In addition to its UFF and Dreiding based terms, the BUFF has additional hydrogen bond terms, specialized protein backbone torsions, and a process for deriving charges for amino acids that is independent of other parameterization. These additional parameters have been fit to ab initio quantum mechanical calculations carried out on model systems.\r\n\r\nValidation studies of peptide trimers demonstrate that the BUFF accurately reproduces the quantum mechanical torsional energies. Several other common, highly parameterized forcefields are also applied to the same tripeptide systems, as well as short \u03b1-helical chains and other model systems in order to make a comparison to the BUFF. These studies show that while the BUFF is universal and can be quickly deployed on new systems, such as unnatural amino acids or metal containing systems, it is also at least as accurate as other commonly employed, but highly parameterized, forcefields. The biological universal forcefield described herein is presented as complementary to the MSC forcefield derived for simulations of DNA and other nucleic acids.", "doi": "10.7907/5kyh-4402", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:11451", "collection": "thesis", "collection_id": "11451", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04092019-114920250", "primary_object_url": { "basename": "Morgan_CS_2000.pdf", "content": "final", "filesize": 45223607, "license": "other", "mime_type": "application/pdf", "url": "/11451/1/Morgan_CS_2000.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Full Sequence Design of an Alpha-Helical Protein and Investigation of the Importance of Helix Dipole and Capping Effects in Helical Protein Design", "author": [ { "family_name": "Morgan", "given_name": "Chantal Smith", "clpid": "Morgan-Chantal-Smith" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Our goal is an objective, quantitative design algorithm based on the\r\nphysical chemical forces which determine protein structure and stability. To\r\nthis end, we have developed a cyclical protein design strategy which utilizes\r\ntheory, computation, and experimentation using a variety of protein systems.\r\nWe address the inverse folding problem using a protein design algorithm\r\nwhich objectively predicts protein sequences which are compatible with a\r\ngiven fold.
\r\n\r\nOur protein design methodology was developed using a variety of\r\nproteins, and therefore should be generalizable to many folds and motifs. To\r\ntest the generalizability and expand the size of proteins we have designed,\r\nengrailed homeodomain (enh), a 51-residue helix-turn-helix motif, was used\r\nas a target motif.
\r\n\r\nA series of design calculations and experiments on the thirty surface\r\npositions of enh were performed to probe the importance of the helix dipole\r\nand capping effects in protein design. Rules for which types of residues were\r\nallowed at the helix termini were introduced systematically, resulting in\r\nprogressively more stable proteins. The first design in the series, which had\r\nno considerations for the helix dipole or capping effects, was shown to have\r\nthe same thermal stability as wild-type enh and the protein with the most\r\nstringent rules has a Tm of 75 \u00b0C, 32\u00b0 higher than wild-type and the first\r\ndesign. Therefore, helix dipole and capping effects have a large impact on\r\nour ability to design stable proteins. The ten core residues of enh were\r\nincluded in the design calculation. The resulting protein, a 29-fold mutant of\r\nwild-type, has a Tm of 81 \u00b0C.
\r\n\r\nThe full sequence design of enh was computed stepwise. The eleven\r\nboundary residues were designed in the context of the surface-core design.\r\nThe resulting protein, a 39-fold mutant of wild-type enh, has a melting\r\ntemperature of 114 \u00b0C and is 4.7 kcal/mol more stable than wild-type. The\r\nstructure of the boundary-surface-core design was solved by NMR\r\ntechniques and found to be in excellent agreement with the target structure.\r\nThe top 10 structure have a backbone root-mean-square standard deviation of\r\n0.45 \u00c5 and the root-mean-square standard deviation between the model\r\nstructure and experimental backbones is 1.25 \u00c5.
\r\n\r\nThe side chain selection algorithm was also extended to the design of\r\npeptides to bind tightly to MHC class I proteins. A circular dichroism\r\nspectrometry assay was developed to determine the peptide dissociation\r\nconstants. Three designed peptides were bound more tightly to the MHC\r\nclass I molecule H-2Kd than known peptides. In addition, an investigation of\r\nthe removal of disulfide bonds from toxin folds is discussed.
\r\n\r\n", "doi": "10.7907/8wn3-cv27", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17645", "collection": "thesis", "collection_id": "17645", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08252025-211023743", "primary_object_url": { "basename": "Gallivan_JP_2000.pdf", "content": "final", "filesize": 71361690, "license": "other", "mime_type": "application/pdf", "url": "/17645/1/Gallivan_JP_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Electrostatic Interactions in Chemistry and Biology", "author": [ { "family_name": "Gallivan", "given_name": "Justin Patrick", "clpid": "Gallivan-Justin-Patrick" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Electrostatic interactions such as hydrogen bonds, salt bridges, and cation-\u03c0 interactions\r\nplay a large role in structural biology. A major goal of this thesis is to build\r\nupon previous studies of the cation-1r interaction to further understand its role in\r\nbiological systems. Put simply, we wish to understand how, when, and why Nature\r\nuses cation-\u03c0 interactions.
\r\n\r\nWe begin by highlighting a cation-1r interaction important in the binding of acetylcholine\r\nto the nicotinic acetylcholine receptor (nAChR). By combining ab initio calculations\r\nand molecular neurobiology, we provide compelling evidence that a cation-\u03c0\r\ninteraction is a major determinant of the recognition of acetylcholine by the nAChR.
\r\n\r\nWe then ask a broader question: To what extent does Nature use cation-\u03c0 interactions\r\nwithin protein structures? By surveying the protein databank, we demonstrate\r\nthat energetically significant cation-\u03c0 interactions are quite common within protein\r\nstructures. To explain why, we ask what advantages cation-\u03c0 interactions have over\r\nother noncovalent interactions commonly found in proteins. Using quantum mechanical\r\ncalculations, we study the strengths of cation-\u03c0 interactions and salt bridges in\r\nboth water and in a range of organic solvents. The results suggest that cation-\u03c0 interactions\r\nmaintain their strength over a wide range of solvents, whereas the strength\r\nof a salt bridge is severely attenuated when it is placed in a high-dielectric solvent.
\r\n\r\nWe then turn our attention to a different type of electrostatic interaction - the interaction\r\nbetween water and hexafluorobenzene. We find that in the gas phase, water\r\nbinds to hexafluorobenzene in a geometry in which the lone pairs of electrons located\r\non the oxygen are directed towards the \u03c0-system of the aromatic. This surprising\r\nresult is easily explained using electrostatics. In addition, we present computational\r\nstudies of the triphenylene\u00b7\u00b7\u00b7perfluorotriphenylene \"supramolecular synthon.\"
\r\n\r\nFinally, we return to the nAChR. A challenge in the study of integral membrane\r\nproteins is determining their transmembrane topology. Here we present a potentially\r\ngeneral method for determining not only the transmembrane topology of a functional\r\nneuroreceptor expressed in a living cell, but also the surface accessibility of individual\r\namino acids.
", "doi": "10.7907/hj9z-mp47", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17638", "collection": "thesis", "collection_id": "17638", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08202025-165630518", "primary_object_url": { "basename": "Chapman_TL_2000.pdf", "content": "final", "filesize": 44922515, "license": "other", "mime_type": "application/pdf", "url": "/17638/1/Chapman_TL_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Biochemical Characterization of Two Cytomegalovirus MHC Class I Homologs", "author": [ { "family_name": "Chapman", "given_name": "Tara Lynn", "clpid": "Chapman-Tara-Lynn" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Strauss", "given_name": "James H.", "clpid": "Strauss-J-H" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Cytomegaloviruses are ubiquitous host-specific pathogens that are capable of causing\r\nlife-long persistent infections in immunocompetent hosts. To maintain this persistence in\r\nthe presence of a functional immune system, both human and murine cytomegaloviruses\r\n(HCMV and MCMV, respectively) encode genes that modulate the host immune\r\nresponse. These genes include the MHC class I homologs UL18 from HCMV and m144\r\nfrom MCMV. The host receptor for UL18 has been identified as LIR-1, a B cell,\r\nmonocyte and dendritic cell inhibitory receptor related to natural killer cell inhibitory\r\nreceptors, whereas the receptor for m144 remains unknown. In order to facilitate\r\nunderstanding of the functions of UL18 and m144 in viral pathogenesis and immune\r\nevasion, we have initiated structure/function analyses of ml 44, UL18 and LIR-1. We\r\nshow that soluble m144 associates with the MHC class I light chain, B2-microglobulin,\r\nbut unlike UL18 and class I MHC proteins, m144 does not associate with endogenous\r\npeptides, presumably due to a large deletion in the peptide binding platform. Using\r\nsoluble versions of UL18, class I MHC molecules and LIR-1, we find that LIR-1 interacts\r\nwith the relatively non-polymorphic a3 domain of class I proteins and the analogous\r\nregion of ULI 8 using its N-terminal immunoglobulin-like domain. Recognition of the a3\r\ndomain, which is relatively non-polymorphic in class I MHC molecules, predicts that\r\nLIR-1 can interact with most or all class I MHC molecules, consistent with previous\r\nobservations that LIR-1 binds a wide range of class I proteins. We also find that LIR-1\r\nbinds UL18 with a > 1000-fold higher affinity than it binds classical and non-classical\r\nclass I MHC proteins, illustrating how a viral protein can effectively compete with host\r\nproteins to subvert the host immune response.", "doi": "10.7907/1jbr-8b50", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17775", "collection": "thesis", "collection_id": "17775", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11252025-213630272", "primary_object_url": { "basename": "Iverson_TM_2000.pdf", "content": "final", "filesize": 81815819, "license": "other", "mime_type": "application/pdf", "url": "/17775/1/Iverson_TM_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Crystallographic Investigations of Respiratory Proteins", "author": [ { "family_name": "Iverson", "given_name": "Tina Michelle", "orcid": "0000-0001-8816-6352", "clpid": "Iverson-Tina-Michelle" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "All organisms require a respiratory process to produce energy. In eukaryotes,\r\nthis process occurs in the mitochondria, and requires a respiratory chain of four integral\r\nmembrane proteins as well as a membrane-soluble quinone pool and cytochrome c. The\r\nrespiratory proteins transfer electrons to oxygen as the terminal electron acceptor with\r\nthe electron transfer coupled to the translocation of protons across the mitochondrial\r\nmembrane. Not all organisms use oxygen as the terminal electron acceptor of their\r\nelectron transport chain. One of the more common alternative electron acceptors is\r\nfumarate, but other common electron acceptors include nitrogen-containing compounds,\r\nthe transformation of which represents an important step in the biological nitrogen cycle.\r\nThis thesis discusses the structural investigations of proteins involved in diverse\r\nrespiratory processes. The crystal structure of the Escherichia coli fumarate reductase,\r\nan integral-membrane enzyme complex involved in anaerobic respiration with fumarate\r\nas the terminal electron acceptor, has been solved. This structure both suggests the\r\nmechanism of the terminal step of anaerobic fumarate respiration and gives a model for\r\nthe function of the homologous protein succinate dehydrogenase from mitochondrial\r\nrespiration. The crystal structure of cytochrome c554 from the chemoautotrophic\r\nnitrifer Nitrosomonas europaea shows a heme-packing motif that may be important in\r\nrespiratory pathways that require the simultaneous transfer of multiple electrons.", "doi": "10.7907/ftcb-ry72", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17666", "collection": "thesis", "collection_id": "17666", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09042025-153842319", "primary_object_url": { "basename": "Shogren-Knaak_MA_2000.pdf", "content": "final", "filesize": 51871805, "license": "other", "mime_type": "application/pdf", "url": "/17666/1/Shogren-Knaak_MA_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Incorporating Function into \u03b2\u03b2\u03b1-Motif Peptide Scaffolds", "author": [ { "family_name": "Shogren-Knaak", "given_name": "Michael Aaron", "orcid": "0000-0001-6526-3977", "clpid": "Shogren-Knaak-Michael-Aaron" } ], "thesis_advisor": [ { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The generation of functional biomolecules constitutes an important and\r\nchallenging goal in bioorganic chemistry. Efforts to incorporate chemical\r\nfunctionality into a \u03b2\u03b2\u03b1-motif (BBA) peptide scaffold are presented. In the course\r\nof this work we have utilized this system to help determine to what extent folded\r\npeptide scaffolds can support chemical function, which strategies are best suited\r\nto the discovery of functional peptides, and what can be learned about biological\r\ncatalysis. To address these issues several different strategies involving the BBA\r\npeptide scaffold have been explored.
\r\n\r\nInitial efforts focused on using the BBA peptide scaffold to modulate the\r\nreactivity of a non-natural pyridoxamine-associated amino acid (Pam). The Pam\r\nresidue was incorporated at three different positions in the BBA peptide, and\r\ndifferent potential general acid and general base residues were included. These\r\npeptides, BP1-BP6, showed improved transamination rates and stereoselectivity\r\nrelative to a model pyridoxamine compound. Additionally, one peptide, BPS,\r\nmaintained many of the secondary and super-secondary structural features of\r\nthe BBA peptide scaffold. A second generation of Pam-containing peptides,\r\npeptides CBP01-CBP18, was constructed based on BPS to investigate the effects\r\nof systematic amino acid changes in the vicinity of the pyridoxamine\r\nfunctionality. These peptides also exhibited enhanced rates of transamination\r\nand stereoselectivity and showed clear trends in stereoselectivity as a function of\r\nthe amino acid substitutions. Finally, peptide systems utilizing other\r\nmechanisms of influencing pyridoxamine-mediated transamination were\r\ninvestigated.
\r\n\r\nSubsequent work, undertaken in collaboration with Kevin McDonnell,\r\nsought to address some of the limitations of the Pam amino acid approach. This\r\neffort focused on developing high-throughput techniques for finding BBA\r\npeptides capable of mediating aldol condensation. A library of over 100,000\r\ndifferent BBA peptides, the CPLB peptides, was designed to incorporate one of\r\nseveral possible basic residues within a core of many potential residues. These\r\npeptides were synthesized and assayed for their ability to sequester a fluorescent\r\nprobe, DMED, which resembles the reaction intermediates in aldol condensation.\r\nThe \"winners\" of this screen exhibited a high degree of sequence similarity,\r\nexhibiting \u03b2-diaminoproprionic acid (Dap) exclusively as the basic residue, and\r\nfavoring aromatic, hydrophobic amino acids as the neighboring residues.\r\nStructural and functional characterization of one of these \"winners,\" peptide\r\nCPLB-A2, was performed. Despite the inclusion of the Dap group within the\r\nhydrophobic core, this peptide appeared to be a monomeric species with a high\r\ndegree of the secondary and super-secondary structure expected of a BBA\r\npeptide. This peptide demonstrated an enhanced ability to sequester DMED\r\nrelative to Dap-containing model peptides and had the capacity to generate an\r\nenamineone reaction intermediate.
\r\n\r\nIn the course of these efforts, techniques were created to aid in the\r\npurification and characterization of N-terminally capped peptides. An affinity\r\npurification method involving a reversible biotin-based capping group was\r\ndeveloped to aid in the isolation of N-terminal glycolyl-capped peptides. This\r\nmethod proved practical for the efficient parallel purification of peptides. A\r\ncapping method involving an \u03b1-chloroacetyl group was developed to generate\r\nN-terminally capped peptides that were not only stable to standard assay\r\nconditions but also capable of being transformed to a form compatible with\r\nEdman sequencing. This method proved to be effective for the identification of\r\nunknown N-terminally capped peptides.
", "doi": "10.7907/wg2w-q908", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:792", "collection": "thesis", "collection_id": "792", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02272009-085501", "primary_object_url": { "basename": "Kossakovski_da_2000.pdf", "content": "final", "filesize": 21161017, "license": "other", "mime_type": "application/pdf", "url": "/792/1/Kossakovski_da_2000.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Scanning probe chemical and topographical microanalysis", "author": [ { "family_name": "Kossakovski", "given_name": "Dmitri A.", "clpid": "Kossakovski-D-A" } ], "thesis_advisor": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The last decade has seen a rapid rise of Scanning Probe Microscopy, SPM, as a prominent and versatile approach for surface studies. SPM instruments are differentiated from the beam-based ones by the fact that they use solid proximal probes for localized analysis. The most commonly used SPM methodology is Atomic Force Microscopy, AFM. In its basic implementation, AFM provides topographical information with nanometer resolution. The most common modifications allow the magnetic, electrostatic, and specific chemical environment to be examined. However, there is no direct way today to perform general chemical analysis with AFM probes.\n\nNear-field Scanning Optical Microscopy, NSOM, is another variation of SPM where sharp tapered optical fibers serve dual purposes, being proximal probes of sample topography, and providing the means for localized light delivery for optical studies with sub-wavelength spatial resolution. Again, NSOM itself does not have a general chemical contrast capability. However, the capability to deliver light to localized area opens the way to a multitude of experiments that can be devised using different aspects of light interaction with the sample.\n\nThis thesis demonstrates several approaches for combined topographical and chemical investigations. Infrared spectroscopy is a sensitive molecular analysis tool. Without scanning proximal probe, IR microscopy has very poor spatial resolution. Enabling methodology for probe fabrication for Near-field Scanning Infrared Microscopy, NSIM, is presented.\n\nThe efforts in combining NSOM with mass spectrometry, which is probably the most general chemical analysis tool, are outlined. We have demonstrated the possibility of simultaneous topographical and molecular imaging.\n\nAnother variation of chemical imaging is the combination of SPM and Laser Induced Breakdown Spectroscopy, LIBS. In this method the elemental composition of samples is obtained by analyzing optical emissions from transient plasma plumes formed by intense laser pulses delivered through fiber probes. We have demonstrated the feasibility of this approach. The instrument that we have developed is an attractive complementary tool for established methods of spatial elemental analysis, such as X-ray Fluorescence. Among its attractive features are operation in ambient conditions, minimal requirements for sample preparation, and ease of use.\n", "doi": "10.7907/at5p-pe62", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17619", "collection": "thesis", "collection_id": "17619", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08132025-181322821", "primary_object_url": { "basename": "Bremer_RE_2000.pdf", "content": "final", "filesize": 48189409, "license": "other", "mime_type": "application/pdf", "url": "/17619/1/Bremer_RE_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Inhibition of DNA Major Groove Binding Proteins by Hairpin Polyamides", "author": [ { "family_name": "Bremer", "given_name": "Ryan Elwood", "clpid": "Bremer-Ryan-Elwood" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind to any predetermined DNA sequence in the human\r\ngenome are potentially useful tools for molecular biology and human medicine.\r\nPolyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) are cell\r\npermeable small molecules that bind DNA according to a set of \"pairing rules\" with\r\naffinities and specificites similar to many naturally occurring DNA binding proteins.\r\nIm/Py polyamides offer a general approach to the chemical regulation of gene expression,\r\nprovided inhibition of DNA binding for a variety of transcription factor families can be\r\nachieved. Polyamides bound in the minor groove have been shown to co-occupy the DNA\r\nhelix with proteins in the major groove. We demonstrate here that polyamides containing\r\na positively charged moiety directed to the DNA backbone can effectively inhibit DNA\r\nbinding by an exclusively major groove protein, potentially by competing with the\r\npositively charged protein side chains for contacts to the negatively charged phosphate\r\nbackbone. The requisite positive patch can be achieved with a naturally derived C-terminal\r\nArg-Pro-Arg tripeptide (Chapter 2) or a simple synthetic diaminoalkyl chain\r\ndelivered from the N-1 of a single pyrrole residue (Chapter 3). The functional repertoire\r\nof poly amides as synthetic ligands for the control of transcription factor binding has been\r\nexpanded to include proteins which bind exclusively in the major groove of DNA. The\r\nbroad targetable sequence repertoire of polyamides, coupled with the ubiquity of\r\nbackbone contacts in protein recognition of DNA, make phosphate neutralization by a\r\npositive patch a promising approach for inhibition of major groove transcription factors.\r\nOther investigations into polyamide:DNA recognition have afforded N=aminoalkylpyrrole-\r\ncontaining polyamides that offer enhanced affinity without\r\ncompromising specificity (Chapter 3), desmethylpyrrole-containing polyamides that\r\nincrease water solubility while retaining subnanomolar affinity (Chapter 4 ), and structural\r\ninsight into the lower affinities observed with Hp-containing polyamides (Chapter 5).", "doi": "10.7907/0rz0-ah38", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17736", "collection": "thesis", "collection_id": "17736", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10292025-210442771", "primary_object_url": { "basename": "Elliott_SJ_2000.pdf", "content": "final", "filesize": 65112980, "license": "other", "mime_type": "application/pdf", "url": "/17736/1/Elliott_SJ_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Copper Centers of Particulate Methane Monooxygenase: Differentiation of C- and E-Clusters", "author": [ { "family_name": "Elliott", "given_name": "Sean Joseph", "orcid": "0000-0003-0096-9551", "clpid": "Elliott-Sean-Joseph" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation investigates the approximately 15 copper ions of pMMO\r\nfrom M. capsulatus by a variety of spectroscopic, biochemical and molecular\r\nbiological techniques. The first chapters describe spectroscopy studies of the C- and\r\nE-clusters of the protein. The pMMO protein is prepared in a number of chemical\r\nstates which access the differentially oxidized of the C- and E-clusters. Investigation\r\nof these states by X-ray Absorption Spectroscopy (XAS) reveals unique oxidation\r\nstates of pMMO copper ions upon anaerobic preparation, and oxidation with air, pure\r\ndioxygen, or hydrocarbon suicide substrate. Structural implications for the copper\r\nions of the protein are discussed, and a model for the ability of acetylene to highly\r\noxidize pMMO is explored. The differentiation of the copper ions of pMMO is\r\nexplored by the use of proteolytic enzymes and chemical treatment with N,N,N',N'-Ethylenediamine-tetraacetic \r\nacid (EDTA), monitoring the ability of pMMO to form a\r\nCu(II)-ferrocyanide adduct upon treatment with ferricyanide and subsequent electron\r\ntransfer. Soluble, copper-binding domains are found, as well as specific centers that\r\nare buried in the transmembrane domain. The inability of an EDTA-treated\r\npreparation to regenerate a fully oxidized form of the protein indicates that the copper\r\nions bound by pMMO do interact with one via electron transfer. The ability to assess\r\nthe potential ligands of the copper ions of pMMO is determined by Electron Spin\r\nEcho Envelop Modulation (ESEEM) spectroscopy, revealing potential interactions\r\nbetween histidine residues and the enzyme active site.
\r\n\r\nSubsequent chapters probe reported characteristic of the pMMO active site,\r\nthought to be located within the PmoA sub-unit. First, the identity of potential\r\nligands for copper ions bound by the PmoA protein is assessed by a homology of\r\npmoA. Next, the role of ion in pMMO is explored by treatment with NO(g), and it is\r\nconcluded that the resulting signals in the g\u22484.0 region of the Electron Paramagnetic\r\nResonance (EPR) spectrum were due to an iron-nitrosyl adduct. The identity of the\r\niron is assigned to adventitious iron bound to pMMO. A final chapter probes the\r\noverall structure and geometry of the active site by examining the regio- and\r\nstereoselectivity of pMMO-mediated hydroxylation and epoxidation chemistry.
", "doi": "10.7907/be24-zt04", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17577", "collection": "thesis", "collection_id": "17577", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07312025-172124033", "primary_object_url": { "basename": "Gordon_DB_2000.pdf", "content": "final", "filesize": 40103293, "license": "other", "mime_type": "application/pdf", "url": "/17577/1/Gordon_DB_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Combinatorial Optimization in Computational Protein Design", "author": [ { "family_name": "Gordon", "given_name": "David Benjamin", "clpid": "Gordon-David-Benjamin" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The central objective of computational protein design is to develop computational\r\ntechniques for selecting amino acid sequences that fold into proteins with desired\r\nstructures and functions. The work described here is directed toward addressing issues\r\nthat arise in the development of computational methods for the design of solvent-exposed\r\nportions of beta-sheets. However, it is also demonstrated that the results of these\r\ninvestigations extend beyond specific secondary structures and in fact provide a means to\r\naddress a broad spectrum of design problems. Computational issues arise from the fact\r\nthat when constructing a representation of protein sequence space for analysis, significant\r\nconcessions must be made with respect to the physical model and the search criteria in\r\norder to ensure that the calculation remains tractable. One of the limiting factors driving\r\nthese concessions is the sheer number of combinations of amino acid identities and\r\nconfigurations that must be evaluated. We have therefore pursued the development and\r\nrefinement of high-performance combinatorial search algorithms in order to better enable\r\nimprovement of computational methods. The consequent algorithmic work consists of\r\nenhancement strategies based on combining optimization methods and instilling within\r\nthem heuristics that manifest specialized knowledge of protein design problems. The\r\nresults are significant performance enhancements for the well-established Dead-End\r\nElimination algorithm, as well as two new algorithmic approaches, dubbed Branch and\r\nTerminate and Hybrid Rotamer Optimization.", "doi": "10.7907/fzxm-d394", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17577", "collection": "thesis", "collection_id": "17577", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07312025-172124033", "primary_object_url": { "basename": "Gordon_DB_2000.pdf", "content": "final", "filesize": 40103293, "license": "other", "mime_type": "application/pdf", "url": "/17577/1/Gordon_DB_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Combinatorial Optimization in Computational Protein Design", "author": [ { "family_name": "Gordon", "given_name": "David Benjamin", "clpid": "Gordon-David-Benjamin" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The central objective of computational protein design is to develop computational\r\ntechniques for selecting amino acid sequences that fold into proteins with desired\r\nstructures and functions. The work described here is directed toward addressing issues\r\nthat arise in the development of computational methods for the design of solvent-exposed\r\nportions of beta-sheets. However, it is also demonstrated that the results of these\r\ninvestigations extend beyond specific secondary structures and in fact provide a means to\r\naddress a broad spectrum of design problems. Computational issues arise from the fact\r\nthat when constructing a representation of protein sequence space for analysis, significant\r\nconcessions must be made with respect to the physical model and the search criteria in\r\norder to ensure that the calculation remains tractable. One of the limiting factors driving\r\nthese concessions is the sheer number of combinations of amino acid identities and\r\nconfigurations that must be evaluated. We have therefore pursued the development and\r\nrefinement of high-performance combinatorial search algorithms in order to better enable\r\nimprovement of computational methods. The consequent algorithmic work consists of\r\nenhancement strategies based on combining optimization methods and instilling within\r\nthem heuristics that manifest specialized knowledge of protein design problems. The\r\nresults are significant performance enhancements for the well-established Dead-End\r\nElimination algorithm, as well as two new algorithmic approaches, dubbed Branch and\r\nTerminate and Hybrid Rotamer Optimization.", "doi": "10.7907/fzxm-d394", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17853", "collection": "thesis", "collection_id": "17853", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02022026-212838189", "primary_object_url": { "basename": "Dmochowski_IJ_2000.pdf", "content": "final", "filesize": 64940562, "license": "other", "mime_type": "application/pdf", "url": "/17853/1/Dmochowski_IJ_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Probing Cytochrome P450 with Sensitizer-Linked Substrates", "author": [ { "family_name": "Dmochowski", "given_name": "Ivan Julian", "orcid": "0000-0001-7162-1347", "clpid": "Dmochowski-Ivan-Julian" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The covalent attachment of the photosensitizer [Ru(bpy)3]2+ to a substrate\r\nconstitutes a powerful new method for probing the steric and electronic properties of\r\nburied enzyme active sites. Particularly important targets are oxygenases (cytochromes\r\nP450) involved in drug metabolism and many disease states. The crystal structure of a\r\nP450:Ru-adamantyl complex reveals that the substrate moiety gains access to the active\r\ncenter via a deep channel and rests above the heme much like the natural substrate\r\ncamphor. This structure also identifies significant P450 conformational changes\r\nassociated with substrate binding for the first time: the channel opens via a 6-\u00c5 loop\r\nmovement. Turnover studies show that activity is not significantly diminished in the\r\nP450:Ru-adamantyl complex. Preliminary light-activated substrate turnover experiments\r\nalso show promise.
\r\n\r\nThe binding of Ru-substrates to P450 was measured by time-resolved\r\nluminescence and UV-vis assays. These molecules specifically recognize submicromolar\r\ncytochrome P450cam in the presence of other heme proteins. In the P450:Ru-substrate\r\nconjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence\r\ndecay. Quantifying the fraction of quenched Ru2+* provides an accurate method for\r\ndetermining dissociation constants. In addition, for the P450:Ru-adamantyl complex,\r\nForster analysis of the energy-transfer kinetics yields a Ru-Fe distance (21 \u00c5) virtually\r\nthe same as that measured in the crystal structure. Similar analysis for other Ru-substrates\r\nof varying length shows a narrow range of Ru-Fe distances, indicating\r\nfavorable association of the {Ru(bpy)3}2+ moiety with the protein. The binding of the \u039b\r\nand \u0394 enantiomers of Ru-C9-Ad to P450 was measured, and the results, KD(\u0394/\u039b) ~2,\r\nsuggest that the bipyridyl ligands interact with aromatic residues at the mouth of the\r\nsubstrate channel. Thus, enantiospecific interactions may be exploited in the design of\r\nenzyme-metallosubstrate conjugates.
\r\n\r\nOxidative and reductive quenching of P450:[Ru-(CH2)n-substrate]2+* conjugates in\r\nsolution triggers the injection of holes and electrons from ruthenium to the heme on submillisecond\r\ntime scales. In order to accelerate electron transfer, conjugated\r\n(perfluorobiphenyl-bridged) Ru-probes were synthesized which bind P450 strongly (KD<\r\n1 \u03bcM). Photoexcitation of conjugated Ru-imidazole-P450 complexes reduces the heme\r\non submicrosecond time scales, opening new avenues for the study of short-lived enzyme\r\nintermediates.
", "doi": "10.7907/89h6-qd11", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17756", "collection": "thesis", "collection_id": "17756", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11112025-205555750", "primary_object_url": { "basename": "Chirik_PJ_2000.pdf", "content": "final", "filesize": 155787462, "license": "other", "mime_type": "application/pdf", "url": "/17756/1/Chirik_PJ_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Ancillary Ligand Effects on Fundamental Transformations in Metallocene Catalyzed Olefin Polymerization", "author": [ { "family_name": "Chirik", "given_name": "Paul James", "orcid": "0000-0001-8473-2898", "clpid": "Chirik-Paul-James" } ], "thesis_advisor": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The preparation of a series of unlinked and ansa-zirconocene dihydride complexes from hydrogenation of the corresponding dimethyl complexes is described. In general, sterically demanding ligands promote formation of monomeric dihydride complexes. The ansa-zirconocene dihydride, meso-[Equation. See abstract in scanned thesis for details], has been characterized by X-ray diffraction. The monomeric, ansa-zirconocene, rac-[Equation. See abstract in scanned thesis for details] undergoes thermal reductive elimination of dihydrogen forming, BpZr(\u03bc2-N2)ZrBp which displays a side-on coordination of the dinitrogen fragment.
\r\n\r\nRates of olefin insertion and \u03b2-hydrogen elimination have been measured for a series of zirconocene and hafnocene dihydride and alkyl hydride complexes. In both cases, increased cyclopentadienyl substitution slows the rate of insertion or elimination, although the former is more sensitive to steric perturbations. From these studies, the transition state for olefin insertion/\u03b2-hydrogen elimination has been established. Equilibration of zirconocene isobutyl hydride complexes with the corresponding normal butyl hydrides has allowed for determination of the relative ground state energies of the two alkyl hydride metallocenes. These data in combination with the activation barriers for \u03b2-hydrogen elimination have allowed for delineation of ground and transition state effects in these processes. Likewise, ancillary ligand effects on the rate of alkyl isomerization have also been examined with a series of isotopically labeled zirconocene alkyl complexes. In general, increasing the substitution on the cyclopentadienyl rings has little effect on the rates of isomerization.
\r\n\r\nThe preparation of ansa-tantalocene olefin-hydride complexes is described. These complexes serve as models for the transition state for olefin insertion in the corresponding group IV metallocene olefin polymerization catalysts. Preparation of the Ci-symmetric ethylene hydride complex, [Equation. See abstract in scanned thesis for details] affords predominantly (-95 %) of one isomer, where the ethylene ligand is coordinated on the side of the wedge away from the isopropyl substituent. Similar results have been obtained with related tantalocene propylene-hydride and styrene-hydride complexes, the latter being characterized by X-ray diffraction. Additionally, several singly and doubly bridged tantalocene trimethyl complexes have been prepared and characterized by X-ray diffraction.
", "doi": "10.7907/2qx2-9753", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17579", "collection": "thesis", "collection_id": "17579", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07312025-184818403", "type": "thesis", "title": "Crystal Structures of ModA from Escherichia coli and Formaldehyde Ferredoxin Oxidoreductase from Pyrococcus furiosus", "author": [ { "family_name": "Hu", "given_name": "Yonglin", "clpid": "Hu-Yonglin" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The crystal structures of two proteins, ModA from Escherichia coli and formaldehyde\r\nferredoxin oxidoreductase from Pyrococcus furiosus, are reported in this paper.
\r\n\r\nModA is a periplasmic molybdate binding protein. Crystals of this protein complexed\r\nwith tungstate or molybdate belong to space groups P3221, with cell dimensions\r\nof a=b=82.6\u00c51, c=81.5\u00c5, \u03b1=\u03b2=90\u00b0, and \u03b3=120\u00b0. The structure of ModA was\r\nsolved by the Single Isomorphous Replacement and Anomalous Scattering method\r\nand refined to 1.75\u00c5 resolution for both molybdate- and tungstate-bound crystal\r\nforms. The Rand free R factors are 16.2% and 20.3%, respectively, for the molybdate-bound\r\nmodel, and 16.3% and 18.6%, respectively, for the tungstate-bound model.\r\nBased on the structural comparisons with other periplasmic binding proteins, such as\r\nsulfate and phosphate binding proteins and ModA from Azotobactor vinelandii, the\r\nstructural bases of the high specificity of ModA for molybdate were identified.
\r\n\r\nP. furiosus formaldehyde ferredoxin oxidoreductase (FOR) was crystallized m\r\nspace group P212121, with cell dimensions a=99.03\u00c5, b=171.10\u00c5, c=179.86\u00c5, and\r\n\u03b1=\u03b2=\u03b3=90\u00b0. Its crystal structure was solved by the molecular replacement method,\r\nand refined to 1.85\u00c5 resolution to an R factor of 17.4%, and free R factor of 22.0%.\r\nComplexes of FOR with glutarate, an inhibitor, and P. furiosus ferredoxin, its physiological\r\nelectron acceptor, were solved and refined to 2.4\u00c5 and 2.15\u00c5 resolution,\r\nrespectively. A structural comparison revealed that FOR may have an enzymatic\r\nmechanism similar to that of Desulfovibrio gigas Mop, an unrelated molybdenum-containing\r\nenzyme. Residues related to the substrate specificity of FOR were identified\r\nbased on the FOR-glutarate interactions. From the arrangement of the redox\r\ncenters in the FOR-ferredoxin complex, an electron transfer pathway between these\r\ntwo partners was proposed.
", "doi": "10.7907/j04r-6905", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:1634", "collection": "thesis", "collection_id": "1634", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05052006-141420", "primary_object_url": { "basename": "White_s_1999.pdf", "content": "final", "filesize": 6843436, "license": "other", "mime_type": "application/pdf", "url": "/1634/1/White_s_1999.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Recognition of All Four Watson-Crick Base Pairs in the Minor Groove of DNA by Synthetic Ligands", "author": [ { "family_name": "White", "given_name": "Sarah", "clpid": "White-Sarah" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The design of synthetic ligands that read the information stored in the DNA double helix has been a long standing goal at the interface of chemistry and biology. Cell-permeable small molecules which target predetermined DNA sequences offer a potential approach for the regulation of gene-expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach is limited to purine tracks and suffers from poor cellular uptake. The subsequent development of pairing rules for minor groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids offers a second code to control sequence specificity. An Im/Py pair distinguishes G\u2022C from C\u2022G and both of these from A\u2022T/T\u2022A base pairs. A Py/Py pair specifies A,T from G,C but does not distinguish A\u2022T from T\u2022A. In order to break this degeneracy, a new aromatic amino acid, 3-hydroxypyrrole (Hp), has been added to the repertoire to test for pairings which discriminate A\u2022T from T\u2022A. We find that replacement of a single hydrogen atom with a hydroxy group in a Hp/Py pairing regulates affinity and specificity by an order of magnitude. By incorporation of a third amino acid, hydroxypyrrole-imidazole-pyrrole polyamides form four ring-pairings (Im/Py, Py/Im, Hp/Py, and Py/Hp) which distinguish all four Watson-Crick base pairs in the minor groove of DNA.", "doi": "10.7907/wxsr-8x37", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:17634", "collection": "thesis", "collection_id": "17634", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08192025-174058689", "primary_object_url": { "basename": "Wang_G_1999.pdf", "content": "final", "filesize": 50451000, "license": "other", "mime_type": "application/pdf", "url": "/17634/1/Wang_G_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Design and Synthesis of Photoreleasable Ubiquinol and its Biologically Active Analogues", "author": [ { "family_name": "Wang", "given_name": "Guangyang", "clpid": "Wang-Guangyang" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Every organism contains a respiratory chain that converts energy from food\r\nmolecules into adenosine triphosphate (ATP) which drives a multitude of biochemical\r\nreactions. A respiratory chain achieves this via a series of integral membrane protein\r\ncomplexes that produce a transmembrane proton gradient as a result of sequential\r\nelectron transfers through these complexes. Ubiquinol and ubiquinol oxidase enzymes are\r\nimportant components of the respiratory chains in most aerobic organisms. The energy\r\nfrom the oxidation of ubiquinol is used to promote the generation of an electrochemical\r\ngradient across the cytoplasmic or mitochondrial membrane for ATP synthesis.
\r\n\r\nStudy of the catalytic mechanisms of ubiquinol oxidase enzymes is very difficult\r\ndue to the structural complexity of these proteins. Since electron input from ubiquinol\r\nplays a very important role in enzyme function, to understand the detailed enzymatic\r\nmechanism of these enzymes, it is critical to understand the kinetics of individual\r\nelectron transfer events in enzymatic turnover. On account of the rapidity of electron\r\ntransfer in these proteins, traditional stopped-flow methods for following the kinetic\r\ncourse of electron transfer reactions are limited, due to the millisecond order of mixing\r\ndead time.
\r\n\r\nPhotochemical initiation of ubiquinol release, a method of circumventing the\r\nmixing limitation, is investigated. The method is based on the photolysis of small\r\norganic protecting groups, or \"cage\" compounds. These cage compounds rendered\r\ninactive quinol-cage complex prior to photolysis by linking to the key functional groups\r\nin ubiquinol or by generating a large steric hindrance that blocks ubiquinol binding.\r\nPhotolysis of the cage-quinol complex rapidly releases the two-electron donor and\r\ntriggers on the enzymatic electron transfer. Such a complex can be used not only in\r\nenzymatic electron transfer study, but also in time-resolved protein conformation change\r\nstudy.
\r\n\r\nThe cage compounds that have been studied for this purpose are based on 3',5'-\r\ndimethoxybenzoin (DMB). Esters of DMB are known to photolyze rapidly (on the sub-nanosecond\r\ntime scale), generating the parent acid and the inert photoproduct 5,7-\r\ndimethoxy-2-phenylbenzofuran. A water-soluble derivative of 3', 5'-dimethoxybenzoin\r\n(DMB),3',5'-bis (carboxylmethoxy)benzoin (BCMB) was linked to the hydroxy group\r\nin ubiquinol via a carbonate linkage. Such a complex has been measured to yield a\r\nphotorelease rate of \r\n990s-1 in detergent solution. Another cage compound, N-hydroxypyridine-\r\n2-thione, was also used to \"cage\" ubiquinol via a carbonate linkage. In\r\nthe presence of efficient hydrogen donor, such as mercaptoethanol, such a complex has\r\nthe potential to photolytically generate ubiquinol in sub-millisecond time scale.
\r\n\r\nA series of ubiquinol analogues were synthesized with a carboxylic acid\r\nfunctionality attached to benzoquinone at different positions. These analogues can be\r\n\"caged\" by BCMB via an ester bond with the potential to trigger electron transfer in sub-microsecond.
\r\n\r\nIn the mean time, efforts have been made to elucidate the controversial photolysis\r\nmechanisms of benzoin compounds by synthetic substitution study and quantum\r\nchemistry calculation. Very efficient synthetic schemes for the preparation of large-size\r\ncombinatorial libraries of benzoins were developed.
", "doi": "10.7907/str8-et07", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:17072", "collection": "thesis", "collection_id": "17072", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03192025-175557925", "primary_object_url": { "basename": "Wagner_PA_1999.pdf", "content": "final", "filesize": 70820925, "license": "other", "mime_type": "application/pdf", "url": "/17072/1/Wagner_PA_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Structural Investigation of Zeolites", "author": [ { "family_name": "Wagner", "given_name": "Paul A.", "clpid": "Wagner-Paul-A" } ], "thesis_advisor": [ { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Microporous materials (including zeolites) that contain molecular-sized pores and\r\ncavities have found wide-spread use in industry as molecular sieves for chemical separations,\r\nas ion-exchangers for detergents and as heterogeneous, shape-selective catalysts. The number\r\nof unique molecular sieve structures discovered over the past few decades has burgeoned and\r\ncurrently is over 121.
\r\n\r\nKnowledge of the crystal structure of these microporous solids can provide important\r\ninsights into their properties that can ultimately lead to the design of desirable materials.\r\nHowever, the structure solution of microporous materials can be challenging because they tend\r\nto form as micron or submicron sized crystals that are too small for single crystal X-ray\r\nanalysis. Thus, the objective of this work is to develop and apply new techniques for solving\r\nthe structures of microporous materials that tend to form micro- and nanocrystals and to utilize\r\nthese structural investigations to gain a more thorough understanding of the zeolite/ organic\r\nstructure directing agent (SDA) interactions that lead to the observed zeolite phase selectivity in\r\ntheir synthesis.
\r\n\r\nIn the absence of single crystal data, structure solution and refinement have typically\r\nrequired the use of powder X-ray data. The difficulty in solving crystal structures from\r\npowder X-ray data is that the three dimensions of information available in a single crystal data\r\nset are collapsed into one dimension (d-spacing) in a powder X-ray data set. If the reflections\r\nin the powder X-ray data are significantly overlapping then solving the crystal structure from\r\nthis data can be extremely difficult. Several techniques are applied here for solving\r\nmicroporous crystal structures from powder X-ray data.
\r\n\r\nThe structure solution of CIT-5 (California Institute of Technology Number 5), a new\r\nhigh-silica molecular sieve synthesized under hydrothermal conditions in the presence of\r\nN(16) methylsparteinium and lithium cations, is obtained though an iterative process of model\r\nbuilding and comparison of the simulated powder X-ray data with the experimental powder X-ray\r\ndata. Rietveld refinement of the synchrotron powder X-ray data supports the symmetry and\r\nspace group assignment for the structure as Pmn21(No.31) with refined unit cell parameters of\r\na=l3.6738(8) \u00c5, b=S.0216(3) \u00c5 and c=25.4883(7) \u00c5 (V=1750.1 \u00c53) and confirms that CIT-5\r\nis the first ordered zeolite to contain one-dimensional extra-large pores circumscribed by\r\n14 tetrahedral-atoms (14 MR).
\r\n\r\nComputational techniques for solving the structures of microcrystals from powder Xray\r\ndata are continuing to increase in sophistication and capability. The crystal structures of\r\ntwo high-silica molecular sieves, SSZ-44 and SSZ-35, are solved using Fourier recycling and\r\nrepresents the first application of this new computational technique for solving novel high-silica\r\nzeolite structures from powder X-ray data. Both materials contain unusual 1-dimensional\r\npores circumscribed by 10 and 18 membered-rings, and are the first high-silica zeolites found\r\nthat possess pores containing greater than 14 membered-rings.
\r\n\r\nElectron diffraction data, obtained from a transmission electron microscope (TEM),\r\nhas inherent advantages over X-ray data for analyzing small crystals due to the stronger\r\ninteraction between the electron beam and matter compared to X-rays. This stronger\r\ninteraction allows a single crystal diffraction data set to be obtained from much smaller\r\ncrystals. Provided that the interaction of the incident electron beam with the crystal is\r\nnearly kinematical direct methods can be used as a powerful tool for obtaining the phase\r\ninformation required to solve the crystal structure.
\r\n\r\nThe development of electron diffraction methods for solving the structure of\r\nnanocrystals is described and the application of this technique to solve the structure of a\r\nlarge-pore, high-silica zeolite, SSZ-48, that contains an occluded organic structure directing\r\nagent is presented. The structure is confirmed by electron diffraction refinement and by\r\nhigh resolution transmission electron microscopy and is found to contain a one-dimensional\r\npore system circumscribed by 12 tetrahedral atoms (12 MR). SSZ-48 is the most complex\r\nthree-dimensional material to be solved at atomic resolution using electron diffraction\r\nmethods and illustrates the power of electron diffraction data for resolving the structures of\r\nmaterials that form crystals too small for standard single crystal X-ray analysis.
\r\n\r\nThese investigations into the structural details of micro- and nanocrystalline\r\nmicroporous materials can be utilized to gain a more thorough understanding of the zeolite/\r\norganic structure directing agent (SDA) interactions that lead to the observed zeolite\r\nsynthesis phase selectivity. Two studies are conducted to probe the relationship between\r\nthe organic structure directing agent and the zeolite framework that is formed from its use.
\r\n\r\nThe first study probes the interaction between the CIT-5 framework and the N(l)-\r\nmethyl-\u03b1-isosparteine SDA I that is found to be a more effective structure directing agent\r\nfor CIT-5 than the diastereomer N(l6)-methylsparteinium II originally used to direct this\r\nnew high-silica zeolite. Molecular modeling calculations reveal that I is capable of forming\r\na greater number of van der Waals interactions with the framework than II thereby\r\nproviding a greater degree of stabilization for the CIT-5 structure as compared to II.
\r\n\r\nFinally, a study into the guest/host interactions between three new zeolite\r\nstructures, SSZ-35, SSZ-36 and SSZ-39 and the 37 organic structure directing agents that\r\nare capable of directing for these zeolites is presented. The size and shape of the organic\r\nSDAs presented in this study are designed in order to obtain novel, open framework\r\nzeolites. The design effort focused on synthesizing large rigid spheroidal SDAs that will\r\npreclude the crystallization of the commonly observed clathrates and straight I-dimensional\r\nchannel system zeolites that result when either small or rigid elongated molecules are\r\nemployed as SDAs. Computational calculations of the organic/inorganic energy of\r\ninteractions provided significant insights into the observed zeolite phase selectivity by the\r\norganic SDAs. The molecular modeling investigations presented here highlight the\r\npotential for developing a rational route to the design of desirable zeolite frameworks.
", "doi": "10.7907/t8qz-qa09", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:2632", "collection": "thesis", "collection_id": "2632", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06172008-140047", "primary_object_url": { "basename": "Yu_b_1999.pdf", "content": "final", "filesize": 6245579, "license": "other", "mime_type": "application/pdf", "url": "/2632/1/Yu_b_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "An engineered mutant of G protein [alpha] subunit that binds xanthine nucleotide and not guanine nucleotide", "author": [ { "family_name": "Yu", "given_name": "Bo", "clpid": "Yu-B" } ], "thesis_advisor": [ { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" } ], "thesis_committee": [ { "family_name": "Simon", "given_name": "Melvin I.", "clpid": "Simon-M-I" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis examines the construction and characterization of mutants of G protein [alpha] subunits that bind xanthine nucleotides, but not guanine nucleotides. G proteins play a critical role in transducing extracellular signals across the cell membrane. The mechanisms of G protein-mediated signal transduction are reviewed in chapter 1.\n\nChapter 2 describes the characterization of the first engineered xanthine nucleotide binding mutant of a G[alpha] subunit, Go[alpha]X (Go[alpha]D273N/Q205L). Go[alpha]X switched nucleotide binding specificity; it bound xanthine nucleotides instead of guanine nucleotides. Go[alpha]X formed an heterotrimer with [beta][gamma] subunits when in the XDP form. Binding of XTP induced a conformational change in Go[alpha]X similar to that of the activated wild-type Go[alpha] and promoted its dissociation from the [beta][gamma] complex.\n\nIn chapter 3, we characterized the receptor interaction of Go[alpha]X. It was able to interact with G protein-coupled receptors effectively; the stimulated m2 muscarinic acetylcholne receptor catalyzed the XTP[gamma]S binding of Go[alpha]X, and the Go[alpha]X[beta][gamma] complex induced the high affinity ligand-binding state in the N-formyl peptide receptor. Interestingly, we found that the empty Go[alpha]X, in the nucleotide-free state, formed a stable complex with receptor and inhibited the activity of Go-coupled receptors in COS-7 cells.\n\nIn chapter 4, we extended this study to two other G proteins. We constructed similar xanthine nucleotide binding mutant proteins in G11[alpha] and G16[alpha] and found that G11[alpha]X (G11[alpha]D277N/Q209L) and G16[alpha]X (G16[alpha]D280N/Q213L) bound XTP[gamma]S and not GTP[gamma]S when expressed in COS-7 cells. Empty G11[alpha] and G16[alpha] mutants also interacted with their cognate receptors and blocked their activity. Similar to Go[alpha]X, both G11[alpha]X and G16[alpha]X retained the receptor specificity of their wild-type proteins and can be used to inhibit subsets of G protein-coupled receptors.\n\nIn chapter 5, we constructed recombinant retroviruses encoding G16[alpha]X, and obtained NIH3T3 cell lines stably expressing the empty G16[alpha] mutants by viral infection. We found that G16[alpha]X blocked the activation of the endogenous thrombin and lysophophatidic acid (LPA) receptors in NIH3T3 cells. These experiments proved that retroviral gene expression can be an effective technique for delivering empty G protein mutants into cells.\n", "doi": "10.7907/K78Q-9K42", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:1532", "collection": "thesis", "collection_id": "1532", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04282006-113514", "primary_object_url": { "basename": "Baird_ee_1999.pdf", "content": "final", "filesize": 22043450, "license": "other", "mime_type": "application/pdf", "url": "/1532/1/Baird_ee_1999.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Solid Phase Synthesis of DNA-Binding Small Molecules", "author": [ { "family_name": "Baird", "given_name": "Eldon Eugene", "orcid": "0009-0009-2775-8704", "clpid": "Baird-Eldon-Eugene" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind to any predetermined DNA sequence in the human genome are potentially useful tools for molecular biology and human medicine. A twenty year research effort led by Dr. Peter B. Dervan at the California Institute of Technology has led to the development of \"pairing rules\" to control rationally the sequence-specificity of polyamides that bind in the DNA minor groove. During the course of my Ph.D. research, methodology was developed for the machine-assisted solid phase synthesis of these DNA-binding polyamides (Chapter 2). The large number of polyamides made available by the solid phase synthetic methodology has greatly accelerated the development of this class of molecules. Polyamides prepared by solid phase synthetic methodology have been used by a variety of collaborators to: extend the targetable binding-site size (Chapter 3), recognize predetermined DNA sequences with subnanomolar affinity (Chapter 6), regulate gene expression in human cells (Chapter 7), and recognize all four base pairs in the DNA minor groove (Chapter 8).", "doi": "10.7907/zky0-ha94", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:1532", "collection": "thesis", "collection_id": "1532", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04282006-113514", "primary_object_url": { "basename": "Baird_ee_1999.pdf", "content": "final", "filesize": 22043450, "license": "other", "mime_type": "application/pdf", "url": "/1532/1/Baird_ee_1999.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Solid Phase Synthesis of DNA-Binding Small Molecules", "author": [ { "family_name": "Baird", "given_name": "Eldon Eugene", "orcid": "0009-0009-2775-8704", "clpid": "Baird-Eldon-Eugene" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind to any predetermined DNA sequence in the human genome are potentially useful tools for molecular biology and human medicine. A twenty year research effort led by Dr. Peter B. Dervan at the California Institute of Technology has led to the development of \"pairing rules\" to control rationally the sequence-specificity of polyamides that bind in the DNA minor groove. During the course of my Ph.D. research, methodology was developed for the machine-assisted solid phase synthesis of these DNA-binding polyamides (Chapter 2). The large number of polyamides made available by the solid phase synthetic methodology has greatly accelerated the development of this class of molecules. Polyamides prepared by solid phase synthetic methodology have been used by a variety of collaborators to: extend the targetable binding-site size (Chapter 3), recognize predetermined DNA sequences with subnanomolar affinity (Chapter 6), regulate gene expression in human cells (Chapter 7), and recognize all four base pairs in the DNA minor groove (Chapter 8).", "doi": "10.7907/zky0-ha94", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:1603", "collection": "thesis", "collection_id": "1603", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05042006-105832", "primary_object_url": { "basename": "Zhao_h_1998.pdf", "content": "final", "filesize": 8776904, "license": "other", "mime_type": "application/pdf", "url": "/1603/1/Zhao_h_1998.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Enzyme Design by Directed Evolution", "author": [ { "family_name": "Zhao", "given_name": "Huimin", "clpid": "Zhao-Huimin" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Directed evolution, inspired by Darwinian evolution in Nature, is an effective approach for protein design. An industrially-important enzyme, subtilisin E, has been chosen as the research target. Important methodologies for directed evolution have been developed, including optimizing the error-prone polymerase chain reaction (PCR) to allow easy and precise control of the mutation rate, optimizing DNA shuffling for high fidelity recombination, and developing three new in vitro recombination methods: random priming recombination (RPR), defined primer recombination (DPR) and staggered extension process (StEP) recombination.
\r\n\r\nUsing these techniques, subtilisin E isolated from the mesophilic organism Bacillus subtilis has been rapidly converted into its thermophilic counterpart (without compromising its activity). After five generations of directed evolution, the resulting variant 5-3H5 is as stable as its naturally-occurring thermostable homolog, thermitase, isolated from the thermophilic organism Thermoactinomyces vulgaris. The half-lives of thermal inactivation at 83\u00b0C of both 5-3H5 and thermitase are 3.5 min. Their temperature optima are 76\u00b0C, 18\u00b0C higher than that of wild type subtilisin E. In addition, 5-3H5 is more active than wild type subtilisin E over the whole range of temperatures. The mutations responsible for the enhanced thermostability were identified and mapped into the structure of subtilisin E. Our findings strongly supports the notion that thermal stability is achieved by the cumulative effect of small improvements at many locations within the protein molecule. Thus, not surprisingly, the pursuit of a 'holy grail' of rules for protein thermostabilization was deemed unsuccessful. However, as demonstrated here, directed evolution is a generally applicable, highly effective approach to increase protein thermostability.
\r\n\r\nThe concepts and techniques developed for directed evolution may also be applied to solving problems associated with molecular evolution in Nature. For example, due to significant sequence divergence, identification of the adaptive mutations, neutral mutations and deleterious mutations in evolutionarily-related proteins is a difficult task. We developed a convenient method to identify functional mutations by gene recombination and sequence analysis of a small sampling of the recombined library exhibiting the evolved behavior. As a demonstration, this approach was used to identify the two thermostable mutations out of ten mutations in a laboratory-evolved thermostable subtilisin E variant.
\r\n", "doi": "10.7907/3bcb-4g98", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:7182", "collection": "thesis", "collection_id": "7182", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08062012-111300229", "primary_object_url": { "basename": "Vaughn_de_1998.pdf", "content": "final", "filesize": 21770345, "license": "other", "mime_type": "application/pdf", "url": "/7182/1/Vaughn_de_1998.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Molecular Mechanism of pH Dependent Antibody Binding: Structure/Function Studies on the Neonatal Fc Receptor", "author": [ { "family_name": "Vaughn", "given_name": "Daniel E.", "clpid": "Vaughn-Daniel-E" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Lester", "given_name": "Henry A.", "orcid": "0000-0002-5470-5255", "clpid": "Lester-H-A" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The work described here is an investigation of the molecular mechanism of pH\r\ndependent immunoglobulin G (IgG) binding by the neonatal Fc receptor (FcRn). FcRn\r\nbinds IgG at acidic, but not alkaline pHs, in two important physiological processes. These\r\nprocesses are the acquisition of passive immunity by the fetus or newborn and protecting\r\nIgG from a default degradative pathway.
\r\n\r\nA biosensor assay is used to characterize the interaction of a soluble form of FcRn\r\nwith IgG. Immobilization of FcRn on the biosensor surface reproduces the high affinity\r\nIgG binding observed for membrane bound FcRn, whereas immobilization of IgG results\r\nin lower affinity binding similar to that of the FcRn/IgG interaction in solution. The\r\nstatistical method of cross-validation is used to show that there are two classes of noninteracting binding sites. The IgG binding interaction is characterized for several mutant FcRns with designed amino acid substitutions. These mutations map the functional IgG\r\nbinding site on FcRn.
\r\n\r\nThe structure of FcRn at an alkaline pH is described. This structure determination\r\nreveals an extensive carbohydrate mediated interaction between the dimer related FeRn\r\nmolecules. The physiological relevance of this interaction is discussed in the context of the FcRn dimerization literature. A further refined structure of FcRn at an acidic pH is\r\ndescribed that includes additional carbohydrate structure. These structures are compared\r\nwith specific attention to the pH dependence of FcRn stability and IgG affinity. Finally, a\r\nmechanism for pH dependent antibody binding to FcRn is proposed based on these structures and the body of structure/function literature concerning this interaction.
", "doi": "10.7907/bzyh-hn75", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:1785", "collection": "thesis", "collection_id": "1785", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05132009-113001", "primary_object_url": { "basename": "Dahiyat_bi_1998.pdf", "content": "final", "filesize": 7405459, "license": "other", "mime_type": "application/pdf", "url": "/1785/1/Dahiyat_bi_1998.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Protein Design Automation : Principles and Practice", "author": [ { "family_name": "Dahiyat", "given_name": "Bassil I.", "clpid": "Dahiyat-Bassil-I" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "We have conceived and implemented a cyclical protein design strategy that couples theory, computation and experimental testing. Our goal is an objective, quantitative design algorithm that is based on the physical properties that determine protein structure and stability and which is not limited to specific folds or motifs. Such a method should escape the lack of generality that has resulted from design approaches based on system-specific heuristics and/or subjective considerations. A critical component of the development of our methods has been their experimental testing and validation. The use of a design cycle coupling theory, computation, and experiment has improved our understanding of the physical chemistry governing protein design and hence enhanced the performance of the design algorithm.\r\n\r\nOur protein design automation algorithm objectively predicts protein sequences likely to achieve a desired fold by using a side-chain selection algorithm that explicitly and quantitatively considers specific side-chain to backbone and side-chain to side-chain interactions. Using a rotamer description of the side chains, we implemented a fast discrete search algorithm based on the Dead End Elimination Theorem to rapidly find the globally optimal sequence in its optimal geometry. We subdivided the sequence selection problem into regions of proteins expected to be dominated by different factors: the tightly packed buried core, the solvent exposed surface, and the boundary between core and surface. We assessed the accuracy of a scoring function or combination of scoring functions by experimentally testing their sequence predictions. Improvements to the scoring function were derived from the experimental data and incorporated into the design algorithm. In this manner, we developed a scoring function for the core of a protein that considers packing interactions and hydrophobic solvation energy. In order to design boundary residues effectively, the usually neglected effect of exposed hydrophobic surface area was addressed. Scoring functions for the design of surface residues were developed that account for hydrogen bonding interactions and secondary structure propensities of amino acids. These potential functions were used to successfully redesign several proteins. The integration of these scoring functions was tested by designing the sequence for an entire protein and solving the NMR solution structure of the designed protein. This work reports the first successful automated design and experimental validation of a novel sequence for an entire protein.", "doi": "10.7907/08j5-w532", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17757", "collection": "thesis", "collection_id": "17757", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11112025-231937152", "primary_object_url": { "basename": "Williamson_JC_1998.pdf", "content": "final", "filesize": 275410410, "license": "other", "mime_type": "application/pdf", "url": "/17757/1/Williamson_JC_1998.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Ultrafast Gas-Phase Electron Diffraction", "author": [ { "family_name": "Williamson", "given_name": "Joseph Charles", "orcid": "0000-0002-3711-1682", "clpid": "Williamson-Joseph-Charles" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The temporal resolution of pump-probe, gas-phase electron diffraction (GED) has\r\nbeen extended to the picosecond time scale, a three order-of-magnitude improvement.\r\nWith such resolution, GED can now be applied to structural studies of fundamental\r\nchemical dynamics, providing complementary information to conventional ps and fs\r\nspectroscopy techniques. This thesis gives a thorough theoretical and experimental\r\ntreatment of ultrafast gas-phase electron diffraction (UGED). Classical Monte-Carlo\r\nsimulations of coherent chemical dynamics were used to demonstrate that the evolution of\r\nmolecular spatial coordinates can be determined with fs GED. Similarly, ps GED can\r\nreveal the structure of short-lived intermediates in kinetic processes. The circular\r\nsymmetry of GED patterns was predicted to break during ps rotational coherences,\r\nrevealing additional structural detail such as bond angles.
\r\n\r\nInstrumentation for UGED was almost entirely home-built. Femtosecond laser\r\npulses were generated in a colliding-pulse, mode-locked ring dye laser and amplified with\r\na four-stage dye cell arrangement pumped by a Nd:YAG laser. The 620-nm output (2 to\r\n3 mJ, 300 fs uncompressed; 30 Hz) was split into pump and probe arms and frequency-doubled.\r\n95% of the laser intensity was focused onto a molecular beam. The remaining\r\n5% was directed onto a back-illuminated 450-A silver cathode, where ultrafast electron\r\npulses were created via the photoelectric effect. The electrons were accelerated with an\r\n18-kV electron gun and focused to a 300-\u03bcm diameter. Space-charge effects forced a\r\ncompromise between electron number density and temporal resolution: streaking\r\nexperiments revealed that a 1-ps pulse contained 1,000 electrons and a 10-ps pulse\r\ncontained 10,000 electrons.
\r\n\r\nThirty centimeters downstream from the exit of the gun, the electrons intersected\r\nthe pump laser at a 90\u00b0 angle, directly underneath the molecular beam orifice. A\r\ntheoretical analysis of the crossed-beam geometry showed that velocity mismatch between\r\nthe pump photons and the probe electrons also affected the temporal resolution of UGED,\r\nmaking a 3-ps contribution. Approximately 10% of the electrons scattered elastically from\r\nsample molecules within the interaction region, and the resulting diffraction pattern was\r\nrecorded with a scintillator / fused fiber optic / image intensifier / charge-coupled device\r\nimaging system housed in a separate vacuum chamber. Single-electron sensitivity across\r\ntwo-dimensions was necessary because of the extremely low electron flux, and the\r\nestimated detective quantum efficiency of the imaging system was better than 0.5.\r\nGround-state GED patterns of CCl4, SF6, CF3I, CH2I2, and C2F4I2 were recorded using ps\r\nelectron pulses. The diffraction data were processed with a software package developed\r\nin the laboratory, and the resulting modified molecular scattering curves agreed well with\r\ntheory. Radial distribution functions were also calculated.
\r\n\r\nTime zero for the pump-probe experiment was identified to within 1 to 2 ps using\r\nphotoionization-induced lensing (PIL) of the unscattered electron beam. The excitation\r\nlaser ionized a small fraction of the molecular beam sample, and a cylindrical coulombic\r\nlens developed within ps as the nascent photoelectrons escaped the interaction region.\r\nThe effects of this lens on the incident 18-ke V electron beam shape was detected by\r\ndirect-bombardment on a charge-coupled device located inside the scattering chamber;\r\nhigh spatial resolution of ~ 15-\u03bcm was necessary to observe PIL-induced changes in the\r\nelectron beam.
\r\n\r\nDiiodomethane was selected as the prototype molecule for the first ultrafast GED\r\ninvestigation. After establishing time zero with PIL, diffraction patterns were recorded at\r\nseveral time steps around t0. The transients showed that approximately 10% of the CH2I2\r\ndissociated into CH2I and an iodine atom following excitation with the 310-nm pump\r\nlaser. The estimated temporal resolution was 5 to 10 ps.
", "doi": "10.7907/8kj6-m794", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17792", "collection": "thesis", "collection_id": "17792", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12102025-233557591", "primary_object_url": { "basename": "Lim_AC_1998.pdf", "content": "final", "filesize": 60742266, "license": "other", "mime_type": "application/pdf", "url": "/17792/1/Lim_AC_1998.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigation of RNA Tertiary Structure and Function by Transition Metal Complexes", "author": [ { "family_name": "Lim", "given_name": "Ai Ching", "clpid": "Lim-Ai-Ching" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenanthrenequinone diimine (phi) complexes of rhodium(III) were employed to\r\nprobe RNA secondary and tertiary structure. These complexes bind via intercalation in\r\nopen major grooves of RNA and upon irradiation promote strand scission. By probing\r\nboth synthetic and natural molecules containing a variety of tertiary motifs, a systematic and\r\npredictive understanding of the factors involved in RNA recognition by these complexes is\r\nsought. The metal complex Rh(phen)2phi3+ (phen = 1,10-phenanthroline) recognizes and\r\ncleaves synthetic triple helices selectively over double helices. The cleavage sites are\r\ndependent upon maximizing overlap between the phi ligand and the basepairs, and\r\nminimizing charge repulsion between the metal complex and protonated bases. These\r\ncleavage sites have proven useful in explaining rhodium complex cleavage in natural\r\nsystems such as tRNAPhe. With these complexes, we also seek to investigate the\r\ndifferences and similarities in RNA and DNA secondary and tertiary folding, by probing\r\nthe tertiary structure of tDNAPhe compared to tRNAPhe. These complexes have elucidated\r\nthe B-form nature of the DNA duplex as well as the tertiary folding of the DNA molecule,\r\nthus shedding light on the feasibility of using DNA analogs of RNA for structural studies.\r\nThese shape selective probes have also been applied to probe the tertiary structure of HIV\r\nand BIV (TAR (trans-activation response) RNAs. \u0394-Rh(phen)2phi3+ binds with high\r\naffinity (Kb= 6.1 \u00b1 1.3 x 105 M-1) and specificity to sites at and across from a bulge\r\nregion which is the recognition element for the binding of the Tat (trans-activating) peptide.\r\nImportantly, the metal complex recognizes an RNA base-triple the formation of which is\r\nnecessary for transactivation. Derivatives of Rh(phen)2phi3+, Rh(MGP)2phi5+(MGP = 4-\r\nguanidylmethy 1-1, 10-phenanthroline) and Rh(GEB)2phi5+ (GEB = 4-(2-guanidylethyl)-4'methy\r\n1-2,2'-bipyridine) where guanidinium moieties have been added to the ancillary\r\nligands of the rhodium complex, show enhanced affinity and selectivity for HIV and BIV\r\nRNA sequences. This is due to the guanidinium moieties mimicking the arginine side\r\nchains on the native Tat peptide, and making non-specific contacts with the phosphate\r\nbackbone of the RNA. However, even without these functionalities, shape-selection,\r\nmatching the shape of the small metal complex to its nucleic acid target, provides sufficient\r\nselective stabilization for RNA site discrimination. Indeed, these complexes compete\r\neffectively with the specific Tat peptides for their binding sites on their respective TAR\r\nRNAs. These complexes therefore employ shape selection to recognize structural\r\nvariations along the RNA polymer which are important for protein recognition. Shape-selective\r\nrecognition could also be applied to the design of novel small molecules to target\r\nnucleic acid sites with high site-selectivity, in the development of molecules to inhibit\r\nprotein recognition, and, potentially, in the design of new chemotherapeutics.", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17541", "collection": "thesis", "collection_id": "17541", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07182025-180545892", "primary_object_url": { "basename": "Lin_SC_1997.pdf", "content": "final", "filesize": 38508661, "license": "other", "mime_type": "application/pdf", "url": "/17541/1/Lin_SC_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Molecular Recognition of DNA by Rhodium(III)-Zinc Finger Peptide Chimeras", "author": [ { "family_name": "Lin", "given_name": "Susanne Chosein", "clpid": "Lin-Susanne-Chosein" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Covalent chimeras of zinc finger peptide domains with\r\nphenanthrenequinone diimine (phi) complexes of rhodium (III) have been\r\ndesigned, synthesized and their DNA recognition characteristics examined. The\r\nrhodium complex binds in the major groove of DNA by intercalation and allows\r\nthe attached peptide to interact with DNA in a sequence-specific manner.\r\nChimeras of [Rh(phi)2(bpy')]3+ (bpy' = 4-(4-carboxybutyl), 4'-methyl-2,2'bipyridine)\r\nand [Rh(phi)2(phen')]3+ (phen' = (5-amidoglutaryl)-1,10-phen-anthroline)\r\nand four different zinc finger peptides (Sp1 finger 2 and 3, ADR1b and\r\nADR1b-Ala) have been successfully synthesized using solid phase coupling\r\nmethodology. Electronic spectroscopy showed the rhodium complex and\r\npeptide to be essentially independent units. A method to successfully fold the\r\npeptide portion of the chimera with zinc has been developed, and 1H HMR\r\nspectroscopy has been used to confirm folding. The resultant chimeras bind\r\ntightly to DNA, and the rhodium intercalator promotes DNA cleavage with\r\nphotoactivation. Analysis of the DNA sites targeted by the chimeras on DNA\r\nrestriction fragments have demonstrated that the peptide can direct new\r\nrecognition. Variations in the rhodium complexes and peptides resulted in\r\ndifferences in specificity as seen by photocleavage. Studies on smaller\r\noligonucleotides containing the recognition sequences have shown the rhodium -\r\nSp1-2 chimera to bind with affinities of 107-108 M-1 for its target sites. Hence,\r\nformation of rhodium(III) - zinc finger chimeras provide a route to establish\r\nhigh affinity DNA binding by a single zinc finger domain. At some sites, the\r\nrhodium complex and zinc finger appeared to bind independently to adjacent\r\nsegments. For the [Rh(phi)2(phen')]3+ - Sp1 - 2 chimeras, a strong high affinity\r\nsite (Ka greater than or equal to 108 M-1) was observed, where it was postulated that the rhodium\r\ncomplex and zinc finger bind to the opposite strands of the GCG binding site in a\r\ncooperative fashion. These rhodium (III) - zinc finger chimeras represent a new\r\nroute to examine the specific interactions of a single zinc finger with DNA in\r\nchemical detail and provide the basis to build a family of sequence-specific DNA\r\nbinding molecules.", "doi": "10.7907/mss5-wf97", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17536", "collection": "thesis", "collection_id": "17536", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07162025-204744164", "primary_object_url": { "basename": "Luo_J-Y_1997.pdf", "content": "final", "filesize": 45859395, "license": "other", "mime_type": "application/pdf", "url": "/17536/1/Luo_J-Y_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stability and Dynamics Studies of Apo-Azurin from Pseudomonas aeruginosa", "author": [ { "family_name": "Luo", "given_name": "Ji-Ye", "clpid": "Luo-Ji-Ye" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The solution stability and dynamics of apo-azurin from Pseudomonas aeruginosa have\r\nbeen studied. First, the unfolding stability was measured for 20 apo-azurin mutants\r\nmutating at T124 in the context of a \u03b2-sheet host-guest environment. The unfolding free\r\nenergy difference was taken as a measure of the relative \u03b2-sheet propensity for the 20\r\nnaturally occurring amino acids. These data, when combined with data from other\r\nexperimental studies and compared to a statistical analysis of the protein structure data base,\r\nstrongly support intrinsic secondary structure preference as the major determinant of \u03b2-sheet\r\npropensity. Particularly, residues Val, Thr, Ile, Phe, and Tyr as a group are intrinsically\r\nfavored for \u03b2-sheet formation, and residues Pro, Gly, and Asp as a group are intrinsically\r\ndisfavored. The relative contribution of the intrinsic propensity and local context to the \u03b2-sheet\r\nformation was further discussed with the results from mutating S34 to Val and Thr.\r\nThe S34T mutant was more stable than the wild-type protein, but the S34V mutant was less\r\nstable. Secondly, partially perturbed states of different forms of azurin at low pH's were\r\nobserved. Apo-azurin at pH 2.9 is a molten globule-like state, while holo-azurin at pH's as\r\nlow as 2.6 is only partially perturbed. Third, backbone 1H and 15N chemical shift\r\nassignments and solution dynamics of apo-azurin were studied. The overall correlation time\r\nof apo-azurin at 30 \u00b0C was determined to be 5.8 ns, and order parameters were mostly at\r\n0.8-0.95. Residues around the Cu(II) binding site exhibited low order parameters and\r\nsignificant 1H chemical shift difference from those of holo-azurin. Last, the osmolyte\r\nstabilizing effect was studied on RNase A using Hydrogen/Deuterium exchange. Two types\r\nof H/D exchange behavior were observed. The H/D exchange rates of type I residues\r\nbecome slower in the presence of high concentrations of glycine, as expected from the\r\nglobal stability change; the H/D exchange rates of type II residues are not affected, however.", "doi": "10.7907/je80-hr40", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17518", "collection": "thesis", "collection_id": "17518", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07112025-210052342", "primary_object_url": { "basename": "Stowell_MHB_1997.pdf", "content": "final", "filesize": 62997604, "license": "other", "mime_type": "application/pdf", "url": "/17518/1/Stowell_MHB_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Ekmageion", "author": [ { "family_name": "Stowell", "given_name": "Michael H. B", "orcid": "0000-0001-7250-1419", "clpid": "Stowell-Michael-H-B" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "orcid": "0000-0001-6547-1469", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Membrane proteins compose roughly 30% of the proteins in a living organism.\r\nFurthermore, they are the essential link between the outside world and the cell. A large\r\nnumber of important processes occur in the biological membranes; these include the\r\nproduction of cellular energy, the transmission of nerve impulses, and the perception of\r\nlight and sound. Knowledge of how these systems are constructed and how they function\r\nis critical to understanding the biological world around us as well as ourselves. The studies\r\npresented herein were aimed at gaining an understanding of such systems through the\r\ncombination of both structural and functional analysis of these systems. The results\r\npresented here are the author's efforts to understand such systems and in the process\r\ndevelop methods and techniques which help others understand such systems or related\r\nones. This thesis is divided into two sections. The first section is devoted solely to the\r\ninvestigation of membrane proteins. An introductory discussion on the structure and\r\nstability of membrane proteins is presented followed by studies on the photosynthetic\r\nreaction center, succinate:quinone oxidoreductase, cytochrome c oxidase, and the\r\ndevelopment of methods for studying rapid electron transfer in ubiquinone:cytochrome-c\r\noxidoreductases and ubiquinol oxidases. The second section is a compilation of theoretical,\r\nmethodological, synthetic, and biophysical studies. These include the development of\r\nMIRAS phasing methods using xenon gas, facile synthetic methods for benzoin\r\ncompounds, development of universal photoreduction compounds, theoretical models for\r\nproton pumping mechanisms in ubiquinol oxidases, and structural studies on a leucine-rich\r\nrepeat variant protein.", "doi": "10.7907/y2zf-he43", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17528", "collection": "thesis", "collection_id": "17528", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07152025-200013414", "primary_object_url": { "basename": "Mines_GA_1997.pdf", "content": "final", "filesize": 47880549, "license": "other", "mime_type": "application/pdf", "url": "/17528/1/Mines_GA_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Cytochrome c : Folding Triggered by Electron Transfer. Rates of Heme Oxidation and Reduction at High Driving Forces", "author": [ { "family_name": "Mines", "given_name": "Gary Alan", "clpid": "Mines-Gary-Alan" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rates of various intramolecular heme oxidations and reductions in a series of\r\nclosely related RuL2(X)(His33)cytochromes c [L = bipyridine or phenanthroline\r\nderivatives; X = imidazole (im) or cyanide (CN-)] have been measured over a freeenergy\r\nrange of 0.54 to 1.89 eV. The driving-force dependence of Fe2+\u2192Ru3+ electron\r\ntransfer (ET) is well described by semiclassical ET theory with a coupling-limited rate\r\n(kmax) of 2.8 x 106 s-1 and a reorganization energy of 0.74 eV. As predicted by theory,\r\nthe rate of an exergonic (-\u0394G\u00b0 = 1.3 eV) heme reduction reaction,\r\n*Ru2+(bpy)2(im)(His)\u2192Fe3+, falls in the inverted region (k = 2.0 x 105 s-1). In contrast,\r\nthe rates of three highly exergonic heme reductions, *Ru2+(phen)2(CN)(His)\u2192Fe3+ (3.1\r\nx 105 s-1; 1.4 eV), Ru+(4,4'-(CONH(C2H5))2-bpy)2(im)(His)\u2192Fe3+ (2.3 x 105 s-1; 1.44\r\neV), and Ru+(phen)2(CN)(His)\u2192Fe3+ (4.5 x 105 s-1; 1.89 eV), are much higher than\r\nexpected for reactions directly to ground-state products. Agreement with theory is\r\ngreatly improved by assuming that an electronically excited ferroheme\r\n(Fe2+\u2192*Fe2+ ~ 1.05 eV) is the initial product in each of these reactions.
\r\n\r\nIn a separate investigation, rates of folding of ferrocytochromes c from horse\r\n(h-cyt c) and yeast (y-cyt c) were measured over a range of denaturant concentrations\r\n(guanidine hydrochloride, GuHCl) and folding free energies (\u0394Gf) using a new ET\r\ntriggering technique. The backbone structures of the two homologs are similar, but y-cyt\r\nc is ~ 15 kJ mol-1 less stable than h-cyt c and is unfolded at concentrations of GuHCl ~ 1.5\r\nM lower than for h-cyt c. Activation free energies exhibit a linear dependence on GuHCl\r\nand \u0394Gf for both proteins, with folding rates decreasing with increasing concentration of\r\nGuHCl (less negative \u0394Gf). At a given denaturant concentration, the folding rates for y-cyt\r\nc are about an order of magnitude slower than those for h-cyt c, but when the folding\r\nfree energies are matched, folding rates of the two homologs are comparable.
", "doi": "10.7907/rtx8-jn96", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17536", "collection": "thesis", "collection_id": "17536", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07162025-204744164", "primary_object_url": { "basename": "Luo_J-Y_1997.pdf", "content": "final", "filesize": 45859395, "license": "other", "mime_type": "application/pdf", "url": "/17536/1/Luo_J-Y_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Stability and Dynamics Studies of Apo-Azurin from Pseudomonas aeruginosa", "author": [ { "family_name": "Luo", "given_name": "Ji-Ye", "clpid": "Luo-Ji-Ye" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The solution stability and dynamics of apo-azurin from Pseudomonas aeruginosa have\r\nbeen studied. First, the unfolding stability was measured for 20 apo-azurin mutants\r\nmutating at T124 in the context of a \u03b2-sheet host-guest environment. The unfolding free\r\nenergy difference was taken as a measure of the relative \u03b2-sheet propensity for the 20\r\nnaturally occurring amino acids. These data, when combined with data from other\r\nexperimental studies and compared to a statistical analysis of the protein structure data base,\r\nstrongly support intrinsic secondary structure preference as the major determinant of \u03b2-sheet\r\npropensity. Particularly, residues Val, Thr, Ile, Phe, and Tyr as a group are intrinsically\r\nfavored for \u03b2-sheet formation, and residues Pro, Gly, and Asp as a group are intrinsically\r\ndisfavored. The relative contribution of the intrinsic propensity and local context to the \u03b2-sheet\r\nformation was further discussed with the results from mutating S34 to Val and Thr.\r\nThe S34T mutant was more stable than the wild-type protein, but the S34V mutant was less\r\nstable. Secondly, partially perturbed states of different forms of azurin at low pH's were\r\nobserved. Apo-azurin at pH 2.9 is a molten globule-like state, while holo-azurin at pH's as\r\nlow as 2.6 is only partially perturbed. Third, backbone 1H and 15N chemical shift\r\nassignments and solution dynamics of apo-azurin were studied. The overall correlation time\r\nof apo-azurin at 30 \u00b0C was determined to be 5.8 ns, and order parameters were mostly at\r\n0.8-0.95. Residues around the Cu(II) binding site exhibited low order parameters and\r\nsignificant 1H chemical shift difference from those of holo-azurin. Last, the osmolyte\r\nstabilizing effect was studied on RNase A using Hydrogen/Deuterium exchange. Two types\r\nof H/D exchange behavior were observed. The H/D exchange rates of type I residues\r\nbecome slower in the presence of high concentrations of glycine, as expected from the\r\nglobal stability change; the H/D exchange rates of type II residues are not affected, however.", "doi": "10.7907/je80-hr40", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:3383", "collection": "thesis", "collection_id": "3383", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09082006-114255", "primary_object_url": { "basename": "Campisi_d_1996.pdf", "content": "final", "filesize": 7329538, "license": "other", "mime_type": "application/pdf", "url": "/3383/1/Campisi_d_1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Transition metal complexes as probes of DNA sequence-dependent structure", "author": [ { "family_name": "Campisi", "given_name": "Donna", "clpid": "Campisi-D" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Different transition metal complexes have been applied in probing variations in the structure of double helical DNA. The following probes, which all bind DNA noncovalently, have been utilized: Ru(phen)3(2)+, Ru(TMP)3(2)+, Rh(phen)2phi3+, Rh(TMP)2phi3+, Rh(dmbpy)2phi3+, Ru(phen)2dppz2+, Ru(bpy)2dppz2+, and Rh(bpy)2dppz3+ (phen = 1,10 phenanthroline; TMP = 3,4,7,8,-tetramethyl- 1,10-phenanthroline; phi = 9,10-phenanthrenequinone diimine; dmbpy = 5,5'-dimethylbipyridyl; bpy = bipyridyl; dppz = dipyrido[3,2-a;2',3'-c]phenazine). The local structure recognized by [Delta]-Rh(phen)2phi3+ has been defined by comparisons of photocleavage data on crystallographically characterized oligonucleotides with their structural parameters. A quantitative correlation has been determined between [Delta]-Rh(phen)2phi3+ photocleavage and extent of openness in the major groove due to differential propeller twisting, or interpurine angle. Therefore, [Delta]-Rh(phen)2phi3+ has been developed as a probe of DNA propeller twisting in solution. Differences in reaction pathway partitioning between enantiomers of Rh(phen)2phi3+ are attributed to differing extent of shape complementarity with DNA binding sites. Rh(TMP)2phi3+ has been explored in probing DNA mismatches in solution. Both [Delta]-Rh(phen)2phi3+ and Rh(TMP)2phi3+ sensitively mark local structural perturbations in an oligonucleotide, arising from substitution of a CG base pair with TG and AG mismatches. Rh(phen)2phi3+ and Ru(TMP)3(2) have also been applied in probing structural variations in the context of a long DNA strand. A C7 stretch is targeted by Ru(TMP)3(2), an A DNA probe and Rh(phen)2phi3+, a B DNA probe. These results indicate this sequence is heteronomous, containing wide major and minor grooves. [Delta]- and [Lambda]-Rh(phen)2phi3+ also discriminate structural differences between bent and nonbent DNA fragments. Variations in metal complex-DNA interactions have also been examined by a gel electrophoretic mobility assay. Intercalator size, hydrophobicity of ancillary ligands, metal complex charge, and chirality all influence the extent of DNA retardation. Taken together, these studies demonstrate that transition metal complexes can be profitably and uniquely applied towards exploring DNA structural heterogeneity.", "doi": "10.7907/kay4-sz63", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:8062", "collection": "thesis", "collection_id": "8062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02042014-091755725", "primary_object_url": { "basename": "Bren_kl_1996.pdf", "content": "final", "filesize": 60833701, "license": "other", "mime_type": "application/pdf", "url": "/8062/1/Bren_kl_1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structurally Engineered Cytochromes c with Novel Ligand-Binding Properties", "author": [ { "family_name": "Bren", "given_name": "Kara Lynne", "orcid": "0000-0002-8082-3634", "clpid": "Bren-Kara-Lynne" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S. c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O2 binding constants ~ 3x greater than those of myoglobin. The resistance of O2-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.
\r\n\r\nUV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH- coordination and that below pH ~6.5, Fe(III)Ala80cyt c is high-spin with His-H2O heme ligation. Significant differences in the pH dependence of the 1H NMR spectra of S. c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.
\r\n\r\n1H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S. c. Fe(III)Ala80cyt c. 82% of the resonances in the 1H NMR spectrum of S. c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 \u00b1 0.11 \u00c5 for the backbone and 1.32 \u00b1 0.14 \u00c5 for all heavy atoms. The solution structure indicates that a tyrosine in the \"distal\" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.
", "doi": "10.7907/D8HZ-E792", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:14040", "collection": "thesis", "collection_id": "14040", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12222020-175451626", "primary_object_url": { "basename": "forman-je_1996.pdf", "content": "final", "filesize": 143036862, "license": "other", "mime_type": "application/pdf", "url": "/14040/1/forman-je_1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Non-Covalent Interactions in Aqueous Media: Molecular Recognition Studies Through Circular Dichroism and Self-Assembly of Discrete Aggregates", "author": [ { "family_name": "Forman", "given_name": "Jonathan Eric", "clpid": "Forman-Jonathan-Eric" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The application of circular dichroism (CD) spectroscopy to the study of molecular recognition phenomena in chiral water-soluble cyclophane hosts is described. The CD method produces results that complement and expand upon previous NMR studies. This includes allowing the measurement of larger binding constants by allowing studies to be carried out at lower concentrations.
\r\n\r\nUsing the excitonic chirality method, these studies have provided a means of assigning the absolute stereochemistry of the ethenoanthracene building blocks used in preparation of the hosts. This information, along with an x-ray structure of one of the cyclophane molecules, has provided important information concerning host structure. The x-ray structure and CD spectral changes observed on guest binding have also served to provide direct experimental evidence for binding conformations of the hosts.
\r\n\r\nThe chiral hosts have been shown to induce CD in achiral chromophoric guests. Analysis of this induced CD using INDO/S and coupled-oscillator calculations has provided valuable information concerning the conformations of the bound guest. These data complement information obtained in NMR studies (D values) and provide additional insights into the important factors that govern the binding event.
\r\n\r\nFinally, preliminary studies of self-assembling systems in aqueous media are reported. These studies employ etheno- and ethanoanthracene based structures designed to form aggregates with well defined order and discrete stoichiometries. These molecules are designed to aggregate through hydrophobic forces. The aggregate is kept from becoming a micelle using polar groups strategically placed to complement one another within the assembling structure.
", "doi": "10.7907/ce8j-qk19", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:8124", "collection": "thesis", "collection_id": "8124", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03112014-150124711", "type": "thesis", "title": "Binding site size limitations of imidazole-pyrrole polyamides for recognition in the minor groove of DNA", "author": [ { "family_name": "Kelly", "given_name": "James J.", "clpid": "Kelly-J-J" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The discovery that the three ring polyamide Im-Py-Py-Dp containing imidazole\r\n(Im) and pyrrole (Py) carboxamides binds the DNA sequence 5'-(A,T)G(A,T)C(A,T)-3'\r\nas an antiparallel dimer offers a new model for the design of ligands for specific\r\nrecognition of sequences in the minor groove containing both G,C and A,T base pairs. In\r\nChapter 2, experiments are described in which the sequential addition of five N-\r\nmethylpyrrolecarboxamides to the imidazole-pyrrole polyamide Im-Py-Py-Dp affords a\r\nseries of six homologous polyamides, Im-(Py)2-7-Dp, that differ in the size of their binding\r\nsite, apparent first order binding affinity, and sequence specificity. These results\r\ndemonstrate that DNA sequences up to nine base pairs in length can be specifically\r\nrecognized by imidazole-pyrrole polyamides containing three to seven rings by 2:1\r\npolyamide-DNA complex formation in the minor groove. Recognition of a nine base pair\r\nsite defines the new lower limit of the binding site size that can be recognized by\r\npolyamides containing exclusively imidazole and pyrrolecarboxamides. The results of this\r\nstudy should provide useful guidelines for the design of new polyamides that bind longer\r\nDNA sites with enhanced affinity and specificity.
\r\n\r\nIn Chapter 3 the design and synthesis of the hairpin polyamide Im-Py-Im-Py-\u03b3-Im-\r\nPy-Im-Py-Dp is described. Quantitative DNase I footprint titration experiments reveal\r\nthat Im-Py-Im-Py-\u03b3-Im-Py-Im-Py-Dp binds six base pair 5'-(A,T)GCGC(A,T)-3'\r\nsequences with 30-fold higher affinity than the unlinked polyamide Im-Py-Im-Py-Dp. The\r\nhairpin polyamide does not discriminate between A\u2022T and T\u2022A at the first and sixth\r\npositions of the binding site as three sites 5'-TGCGCT-3', 5'-TGCGCA-3', and 5 'AGCGCT-\r\n3' are bound with similar affinity. However, Im-Py-Im-Py-\u03b3-Im-Py-Im-PyDp\r\nis specific for and discriminates between G\u2022C and C\u2022G base pairs in the 5'-GCGC-3'\r\ncore as evidenced by lower affinities for the mismatched sites 5'-AACGCA-3', 5'-\r\nTGCGTT-3', 5'-TGCGGT-3', and 5'-ACCGCT-3'.
\r\n\r\nIn Chapter 4, experiments are described in which a kinetically stable hexa-aza\r\nSchiff base La3+ complex is covalently attached to a Tat(49-72) peptide which has been\r\nshown to bind the HIV-1 TAR RNA sequence. Although these metallo-peptides cleave\r\nTAR site-specifically in the hexanucleotide loop to afford products consistent with\r\nhydrolysis, a series of control experiments suggests that the observed cleavage is not\r\ncaused by a sequence-specifically bound Tat(49-72)-La(L)3+ peptide.
", "doi": "10.7907/jc2t-pr07", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:11901", "collection": "thesis", "collection_id": "11901", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11072019-170728389", "primary_object_url": { "basename": "figl-a-1996.pdf", "content": "final", "filesize": 4412867, "license": "other", "mime_type": "application/pdf", "url": "/11901/1/figl-a-1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-Function Analysis of the \u03b2 Subunit of Neuronal Nicotinic Acetylcholine Receptors", "author": [ { "family_name": "Figl", "given_name": "Antonio", "clpid": "Figl-Antonio" } ], "thesis_advisor": [ { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nicotinic receptors belong to the superfamily of ligand-gated ion channels. Since evidence was rapidly accumulating implicating the non-\u03b1 subunits in ligand-binding events, we decided to investigate eventual contributions of the neuronal \u03b2 subunit to these events by performing a series of increasingly detailed experiments on a series of chimeric \u03b2 subunits. In the first set of experiments, we constructed a variety of chimeric \u03b2 subunits consisting of NH2-terminal neuronal \u03b24 sequences and COOH-terminal \u03b22 sequences and expressed them with the \u03b13 subunit in Xenopus oocytes. The results showed that (a) two residues in the extracellular domain of chimeric \u03b24\u2022\u03b22 subunits (108\u03b22Phe\u2194\u03b24Val, 110\u03b22Ser\u2194\u03b24Thr) account for much of the relative cytisine sensitivity; and (b) four extracellular residues of chimeric \u03b24\u2022\u03b22 subunits (112\u03b22Ala\u2194\u03b24Val, 113\u03b22Val\u2194\u03b24Ile and 115\u03b22Ser\u2194\u03b24Arg, 116\u03b22Tyr\u2194\u03b24Ser) account for most of the relative tetramethylammonium sensitivity.
\r\n\r\nEncouraged by the above results, we continued our experiments with additional chimeras of the \u03b22 and \u03b24 neuronal nicotinic subunits to locate regions that contribute to differences between the acetylcholine dose-response relationships of \u03b13\u03b22 and \u03b13\u03b24 receptors. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other, suggesting that amino acids within the first 120 residues of \u03b22 and the corresponding region of \u03b24 contribute to an agonist binding site that bridges the \u03b1 and \u03b2 subunits in neuronal nicotinic receptors.
\r\n\r\nSince the EC50 phenotypes caused by the \u03b22 and \u03b24 subunits could be due to a difference in gating or binding properties, we attempted to unravel this question by performing voltage-jump relaxations for the series of neuronal nicotinic acetylcholine receptors we constructed previously. The chimeric \u03b24/\u03b22 subunits showed a transition in the concentration dependence of the relaxation rate constants in the region between residues 94 and 109, analogous to our previous observation with steady-state dose-response relationships. The data reinforce previous conclusions that the region between residues 94 and 109 on the \u03b2 subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.
", "doi": "10.7907/e390-k051", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:11900", "collection": "thesis", "collection_id": "11900", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11062019-175229018", "primary_object_url": { "basename": "day-mw-1996.pdf", "content": "final", "filesize": 8964138, "license": "other", "mime_type": "application/pdf", "url": "/11900/1/day-mw-1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "X-Ray Crystallographic Studies on Electron Transfer Proteins; Rubredoxin from Pyrococcus furiosus, Nitrogenase MoFe from Azotobacter vinelandii and Ru(2,2'-bppy)\u2082(imd)His83 Azurin from Pseudomonas aeruginosa", "author": [ { "family_name": "Day", "given_name": "Michael W.", "clpid": "Day-Michael-W" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Marsh", "given_name": "Richard Edward", "clpid": "Marsh-R-E" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The x-ray crystal structure of the oxidized and the reduced forms rubredoxin from Pyrococcus furiosus, a hyperthermophilic marine Archae, have been solved by molecular replacement and refined by the method of restarined least squares to a maximum resolution of 1.1 \u00c5 for the oxidized form and 1.5 \u00c5 for the reduced form. The oxidized form of the protein crystallizes in the orthorhombic space group P 212121 with unit cell dimensions of a = 33.8 \u00c5, b = 34.6 \u00c5, c = 43.4 \u00c5 and V = 50,755 \u00c53. The reduced form crystallizes in the same space group with the nearly identical unit cell dimensions of a = 33.8 \u00c5, b = 34.5 \u00c5, c = 43.2 \u00c5 and V = 50,375 \u00c53. Data on both forms was collected at -161\u00b0C. Three refinement packages were used in the refinement and the results from each arc discussed as are the possible determinants of the thermal stability. Refinement of the oxidized form (414 protein atoms and 104 solvent oxygens) with TNT or XPLOR resulted in a crystallographic residual of approximately 17% and a model with rms deviations of bond distances and angles from target values of approximately 0.015 \u00c5 and 2.5\u00b0 respectively. Refinement of the oxidized form with SHELXL-93 resulted in a model with 132 solvent oxygens and an R = 13.9% (Rfree = 17.2%) and GOF = 1.08. The rms deviation from the target values for bond distance and angles are 0.014 \u00c5 and 1.75\u00b0 respectively. Refinement of the reduced form with TNT (110 solvent oxygens) or SHELXL-93 (including 130 solvent oxygen atoms) results in an R-factor of approximately 17% and the geometry of the model deviates from the target values by approximate rms values of 0.022 \u00c5 for the bond distances and 3.0\u00b0 for the bond angles.
\r\n\r\nThe x-ray crystal structure of the MoFe nitrogenasc protein from Azotobcacter vinelandii has been refined against data collected at the Stanford Synchrotron Radiation Laboratory (SSRL). The data extends to a maximum resolution of 2.2 \u00c5 and two packages were used in the restrained least squares refinement. Refinement of the model (including 625 solvent oxygens) with TNT or XPLOR yielded a crystallographic residual of less than 18% and a model with bond distances and angles deviate from the target values by rms values of 0.02 \u00c5 and 2.5\u00b0 respectively.
\r\n\r\nThe x-ray crystal structure of the Ru(2,2'-bppy)2(imd)His83 azurin from Pseudomonas aeruginosa has been solved and refined by the method of restrained least squares to a limiting resolution of 2.5 \u00c5. The labeled protein crystalizes in the monoclinic space group C 2 with a = 100.6 \u00c5, b = 35.4 \u00c5, c = 74.7 \u00c5, b = 106.5\u00b0 V = 255,069 \u00c53 and Z = 8. Data was collected at -161\u00b0C to a maximum resolution of 2.3 \u00c5 yielding a data set that is 82% complete containing 11,083 reflections. Refinement in TNT (including 150 solvent oxygen atoms) resulted in an R-factor of 17.3% with rms deviations in the model bond distances and angles from ideal values of 0.026 \u00c5 and 3.09\u00b0 respectively.
", "doi": "10.7907/Y70P-TJ52", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:17860", "collection": "thesis", "collection_id": "17860", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02042026-232356481", "primary_object_url": { "basename": "Musser_SM_1996.pdf", "content": "final", "filesize": 71618887, "license": "other", "mime_type": "application/pdf", "url": "/17860/1/Musser_SM_1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Proton Translocation Mechanisms of the Cytochrome bo\u2083-Type Ubiquinol Oxidase Complex and the Mitochondrial Cytochrome c Oxidase Complex", "author": [ { "family_name": "Musser", "given_name": "Siegfried M.", "orcid": "0000-0002-7793-2557", "clpid": "Musser-Siegfried-M" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Every organism contains a respiratory chain that enables it to convert the energy\r\nobtained from food molecules into adenosine triphosphate (ATP), the universal energy\r\ncurrency which drives a multitude of life-giving biochemical reactions. A typical\r\nrespiratory chain contains a series of integral membrane protein complexes that produce a\r\ntransmembrane proton gradient as a result of sequential electron transfers through these\r\ncomplexes. The energy stored in this proton gradient, a biological battery, is utilized to\r\nsynthesize ATP. In the oxygenic respiratory process functioning in mitochondria,\r\ndioxygen is fully reduced to water by the cytochrome c oxidase (CcO) complex. This\r\nmolecular machine couples the highly exergonic reduction of dioxygen to the pumping of\r\nprotons against a transmembrane electrochemical gradient. Another structurally similar\r\nenzyme, the cytochrome bo3 complex from Escherichia coli, catalyzes the reduction of\r\ndioxygen to water using the same heme-copper dioxygen activating site and also\r\ncatalyzes the translocation of protons. However, whereas the electron donor for the CcO\r\ncomplex is the water-soluble one-electron carrier cytochrome c, two-electron-donating\r\nubiquinol molecules within the lipid bilayer input electrons into the cytochrome bo3\r\ncomplex. The dramatically different electron input mechanisms dictated by these\r\nelectron-donating substrates led to the hypothesis that the proton translocation\r\nmachineries for these two terminal oxidases are completely disparate. Based on the\r\navailable data, it is concluded that the cytochrome bo3 complex translocates protons via a\r\nquinone(quinol)-loop (Q(H2)-loop) mechanism. In a Q(H2)-loop proton translocation\r\nmechanism, proton uptake/release follows electron input to/output from two Q(H2)\r\nbinding sites and protein conformational cycling is not required. On the other hand, the\r\nmore complicated redox-linked proton pump of the CcO complex functions by forcing\r\nprotons through the protein matrix as a result of a series of conformational\r\nrearrangements and is successful only through careful control of both electron and proton\r\ntransfer reactions. This thesis focuses on deciphering the proton translocation capabilities\r\nof these two enzymes and contrasts the simplicity of a Q(H2)-loop mechanism with the\r\nmore evolutionarily advanced CcO proton pump.", "doi": "10.7907/nw3n-jd48", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:3935", "collection": "thesis", "collection_id": "3935", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10052007-085601", "primary_object_url": { "basename": "He_q_1995.pdf", "content": "final", "filesize": 5532383, "license": "other", "mime_type": "application/pdf", "url": "/3935/1/He_q_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Cytochrome c oxidase : studies of electron input and intramolecular electron transfer", "author": [ { "family_name": "He", "given_name": "Qizhi", "clpid": "He-Qizhi" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nCytochrome [...] oxidase is the terminal enzyme of the electron transport chain in mitochondria. This enzyme catalyzes the transfer of electrons from ferrocytochrome [...] to dioxygen and reduces it to water. Concomitant with electron transfer and dioxygen reduction, cytochrome [...] oxidase pumps protons from the matrix side to the cytosolic side of the inner membrane, contributing to the formation of a transmembrane electrochemical gradient. As such electron transfer plays a central role in the function of the enzyme. This thesis investigated the input of electrons into the enzyme, the flow of electrons among redox-active metal centers in the enzyme as well as conformational changes associated with the redox state changes of the protein.\r\n\r\nDuring electron transport, cytochrome [...] diffuses to a binding site or sites on cytochrome [...] oxidase. Extensive chemical modification studies have indicated that seven highly conserved lysine amino acids surrounding the heme crevice of cytochrome [...] are involved in the formation of an electrostatic complex with cytochrome [...] oxidase. Lysine 86 in cytochrome [...] is believed to be crucial in the binding of cytochrome [...] with cytochrome [...] oxidase. In order to study the effect of binding orientation between cytochrome [...] and cytochrome [...] oxidase with respect to the intracomplex electron transfer, we modified cytochrome [...] with ruthenium bis(bipyridine) dicarboxybipyridine at lysine 86 (Ru-86-cytochrome [...]). Our results showed that upon laser excitation of the preformed complex of Ru-86-cytochrome [...] and cytochrome [...] oxidase, electron was transferred rapidly from the ruthenium group to the ferric heme of cytochrome [...]; subsequently to the cytochrome [...] oxidase. The observed intracomplex rate constants for the oxidation of cytochrome [...] are found to be biphasic with magnitudes of 560 [...] for one phase and 114 [...] for the other phase. The rate constant for the reduction of cytochrome [...] in cytochrome [...] oxidase is 2.3 x [...]. No reduction of [...] was observed at 830 nm. Although the observed rate constants for the oxidation of cytochrome [...] are slow, we believe there is a fast kinetic phase for this process beyond the resolution capability of the instrument. Apparently, the bulky ruthenated moiety on cytochrome [...] alters the binding orientation of cytochrome [...] with cytochrome [...] oxidase. As a result, cytochrome [...] preferentially transfers electron directly to cytochrome [...] rather than to [...]. The intracomplex electron transfer rate also exhibited ionic strength dependence as expected.\r\n\r\nInternal electron transfer in cytochrome [...] oxidase was investigated by photolysis of CO-bound mixed-valence form of the enzyme. Upon CO photodissociation, ferrocytochrome [...] was generated in less than 0.1 [...]sec, and a subset of the reduced cytochrome [...] was reoxidized with biphasic rate constants of [...] = 1.0 x [...] and [...] = 7.8 x [...]. Reduction of cytochrome [...] was also observed with biphasic rate constants of [...] = 1.6 x [...] and [...] = 9 x [...]. The stoichiometry of oxidized cytochrome [...] to reduced cytochrome [...] was found to be 1:1. No apparent electron transfer to [...] was observed at 830 nm. These results indicate that there is a significant electron reequilibration only between cytochrome [...] and cytochrome [...] upon photodissociation of the CO-bound mixed-valence enzyme.\r\n\r\nThe nature of zinc in cytochrome [...] oxidase was investigated by depletion of zinc with mercuric chloride. The removal of zinc does not alter the steady-state and transient electron transfer activities of the enzyme. The study indicates that zinc plays a structural role in the enzyme by serving as a bridge between subunit VIa and VIb. Based on indications that there is allosteric interaction companying redox state changes of cytochrome [...] oxidase, we probed the protein matrix for conformational changes using a fluorescence label on the enzyme. As a result of our findings, we concluded that there is a redox-linked conformational change in cytochrome [...] oxidase.\r\n", "doi": "10.7907/ve6b-7z16", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:3628", "collection": "thesis", "collection_id": "3628", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09182007-090157", "primary_object_url": { "basename": "Cohen_sb_1995.pdf", "content": "final", "filesize": 7121593, "license": "other", "mime_type": "application/pdf", "url": "/3628/1/Cohen_sb_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Mechanistic studies of the natural DNA-cleaving agents neocarzinostatin chromophore, calicheamicin [gamma]1, and dynemicin A", "author": [ { "family_name": "Cohen", "given_name": "Scott B.", "clpid": "Cohen-S-B" } ], "thesis_advisor": [ { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "thesis_committee": [ { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Carreira", "given_name": "Erick Moran", "clpid": "Carreira-E-M" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\nExperiments establishing the intermediacy of the cumulene derived from neocarzinostatin chromophore are described. It is shown that [...]95% of DNA cleavage arises via the cumulene. The sequence specificity and efficiency of DNA cleavage by externally generated cumulene are identical to that by the cumulene formed in situ, supporting the proposal that the cumulene determines the sequence specificity of DNA cleavage. It is shown that DNA and a water-soluble cyclohexadiene derivative are equally effective in trapping of the biradical intermediate at concentrations of 5 mM and 1 M, respectively, supporting the idea that the biradical must be generated as a DNA-bound species to induce DNA cleavage.\r\n\r\nThe reaction of calicheamicin [...] with glutathione has been studied in the presence of DNA and is shown to produce all four products arising from S-S bond exchange. The calicheamicin-glutathione disulfide is formed as the major product of this reaction, and is shown to be 2-3 orders of magnitude less reactive toward glutathione than is calicheamicin [...]. The rate of DNA cleavage by calicheamicin [...] is essentially independent of the concentration of DNA, while the rate of DNA cleavage by the calicheamicin-glutathione disulfide is inversely proportional to the concentration of DNA. The data support the hypothesis that calicheamicin [...] undergoes thiol activation as a DNA-bound species, while the calicheamicin-glutathione disulfide is activated free in solution.\r\n\r\nBinding constants of dynemicin A and synthetic analogs to DNA show that the two E-ring hydroxyls of the anthraquinone contribute approximately 2.7 kcal/mol binding energy, and that neutralization of the negatively-charged carboxylate stabilizes the drug DNA binding complex by ~3 kcal/mol. Dynemicin A and the synthetic analogs display an inverse rate dependence on the concentration of DNA, supporting the proposal that these drugs must dissociate from DNA prior to chemical activation.\r\n", "doi": "10.7907/ZJH6-AC74", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:4213", "collection": "thesis", "collection_id": "4213", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10222007-132632", "primary_object_url": { "basename": "Sardesai_ny_1995.pdf", "content": "final", "filesize": 17895094, "license": "other", "mime_type": "application/pdf", "url": "/4213/1/Sardesai_ny_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Rhodium intercalators as novel peptide delivery systems to the major groove of DNA : towards the design of artificial repressors", "author": [ { "family_name": "Sardesai", "given_name": "Niranjan Y.", "clpid": "Sardesai-N-Y" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nPhenanthrenequinone diimine (phi) complexes of rhodium(III) bearing tethered peptides have been designed to serve as metallointercalating anchors to deliver peptide side chain functionalities for DNA recognition in the major groove. Metal-peptide \ncomplexes containing 11-15 amino acid residues were prepared using two complementary synthetic strategies: by direct coupling of a pendant carboxylate on the coordinatively saturated rhodium complex, [...] (phen'=5-amidoglutaryl-1, 10-phenanthroline), to the N-terminus of a resin-bound peptide in a manner analogous to the chain-elongation step in solid phase peptide synthesis; or by coupling phen' containing the pendant carboxylate to the resin-bound peptide, followed by coordination of [...] to the bidentate chelator attached to the peptide. With coordination complexes which are stable to peptide deprotection and cleavage conditions from the resin, the solid phase synthetic strategies prove convenient to apply. The metal-peptide complexes have been characterized by amino acid analysis, electronic spectroscopy, circular dichroism and mass spectrometry, where a novel pattern of peptide fragmentation facilitates the detailed sequence analysis of the appended peptide. All the metal-peptide complexes bind and, with photoactivation, cleave DNA with evidence of major groove chemistry. Significantly, the DNA site-specificity is seen to depend on the peptide side-chain functional groups. In one series, a single glutamate at position 10 is found to be essential in directing DNA site-recognition to the sequence 5'-CCA-3'. Methylation of the glutamate side chain or single ElOQ, E1OD, E1OA mutations abolish this selectivity. The glutamate is essential to maintain [...]-helicity in the peptide and make base specific contacts, thereby providing a glutamate switch for site-specific DNA recognition. A second series, based on the recognition helix of the phage 434 repressor, reproduces operator binding. Photocleavage and MPE-Fe footprint analysis indicates that these metal-peptide complexes bind to the 5'-ACAA-3' operator sequences as monomers at\n10 nM concentration and differentiate between operator site variants. These studies represent a new strategy to create an array of metal-peptide complexes with differing sequence specificity for DNA and suggest a route to the construction of small molecules that function as artificial repressors.\n", "doi": "10.7907/0v7r-fp28", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:4111", "collection": "thesis", "collection_id": "4111", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10162007-094617", "primary_object_url": { "basename": "Kim_kh_1995.pdf", "content": "final", "filesize": 7511901, "license": "other", "mime_type": "application/pdf", "url": "/4111/1/Kim_kh_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Crystallographic structure determination of neocarzinostatin, an antitumor protein-chromophore complex", "author": [ { "family_name": "Kim", "given_name": "Kyoung-Hee", "clpid": "Kim-K" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nCrystal structures of the protein-chromophore complex and the apoprotein form of the natural antitumor antibiotic neocarzinostatin (NCS) have been determined at 1.8 [...] resolution. NCS is composed of a labile chromophore component with DNA-cleaving activity and a protein component that binds and stabilizes the chromophore. The NCS protein consists of the large domain of a seven-stranded [beta] barrel and the small domain. The chromophore is bound noncovalently in a pocket between the two domains, primarily through van der Waals contacts with many nonpolar residues. [...] and [...] contact the two [pi] faces of the nine-membered enediyne ring through an edge of each benzene ring. [...] additionally contacts the aminosugar group through its [pi] face. The positioning of [...] represents the most significant difference between the holo- and apo-protein structures; in apo-NCS, [...] rotates to a more solvent-exposed position. The epoxide and C12 (the site of nucleophilic thiol addition during activation of the chromophore) are sequestered from the solvent, which likely contribute to the stability of the chromophore in holo-NCS. The amino group of the chromophore is oriented above C12 at a distance approximately the van der Waals diameter of a sulfur atom, supporting the idea that this group plays a role in the thiol activation mechanism. While the basic protein structure is conserved among at least five chromoprotein antibiotics, [...] is unique to NCS, perhaps accounting for the binding specificity of neocarzinostatin for its chromophore.\n\nWe have systematically investigated the effects of parameters in the molecular replacement methods on NCS using three different programs. The model with side chains of conserved residues and the maximum vector length or the integration radius of about half the diameter of the molecule, gave the best result. The low resolution limit of 15 [...] was needed for the rotation function calculation in both X-PLOR and AMoRe. The program MERLOT failed to give molecular replacement solutions in this case.\n", "doi": "10.7907/6nef-8t49", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:7722", "collection": "thesis", "collection_id": "7722", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05172013-114618257", "primary_object_url": { "basename": "Ross 1994.pdf", "content": "final", "filesize": 24975103, "license": "other", "mime_type": "application/pdf", "url": "/7722/1/Ross 1994.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Insights into the Mechanism of Human Erythrocyte Hexose Transport: a Transferred NOE Study of Glucose Binding to GLUT1", "author": [ { "family_name": "Ross", "given_name": "Scott Alan", "clpid": "Ross-Scott-Alan" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Weitekamp", "given_name": "Daniel P.", "orcid": "0000-0003-0079-8000", "clpid": "Weitekamp-D-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This study examines binding of \u03b1- and \u03b2-D-glucose in their equilibrium mixture to the glucose transporter (GLUT1) in human erythrocyte membrane preparations by an ^1H NMR method, the transferred NOE (TRNOE). This method is shown theoretically and experimentally to be a sensitive probe of weak ligand-macromolecule interactions. The TRNOEs observed are shown to arise solely from glucose binding to GLUT1. Sites at both membrane faces contribute to the TRNOEs. Binding curves obtained are consistent with a homogeneous class of sugar sites, with an apparent KD which varies (from ~30 mM to ~70 mM for both anomers) depending on the membrane preparation examined. Preparations with a higher proportion of the cytoplasmic membrane face exposed to bulk solution yield higher apparent KKDs. The glucose transport inhibitor cytochalasin B essentially eliminates the TRNOE. Nonlinearity was found in the dependence on sugar concentration of the apparent inhibition constant for cytochalasin B reversal of the TRNOE observed in the \u03b1 anomer (and probably the \u03b2 anomer); such nonlinearity implies the existence of ternary complexes of sugar, inhibitor and transporter. The inhibition results furthermore imply the presence of a class of relatively high-affinity (KD < 2mM) sugar sites specific for the \u03b1 anomer which do not contribute to NMR-observable binding. The presence of two classes of sugar-sensitive cytochalasin B sites is also indicated. These results are compared with predictions of the alternating conformer model of glucose transport. Variation of apparent KD in the NMR-observable sites, the formation of ternary complexes and the presence of an anomer-specific site are shown to be inconsistent with this model. An alternate model is developed which reconciles these results with the known transport behavior of GLUT1. In this model, the transporter possesses (at minimum) three classes of sugar sites: (i) transport sites, which are alternately exposed to the cytoplasmic or the extracellular compartment, but never to both simultaneously, (ii) a class of sites (probably relatively low-affinity) which are confined to one compartment, and (iii) the high-affinity \u03b1 anomer-specific sites, which are confined to the cytoplasmic compartment.\r\n", "doi": "10.7907/nb45-1b79", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7664", "collection": "thesis", "collection_id": "7664", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05032013-161139911", "type": "thesis", "title": "A biochemical and structural characterization of Drosophila neuroglian", "author": [ { "family_name": "Huber", "given_name": "Andrew Henry", "clpid": "Huber-Andrew-Henry" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A number of cell-cell interactions in the nervous system are mediated by\r\nimmunoglobulin gene superfamily members. For example, neuroglian, a homophilic\r\nneural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C-\r\n2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats.\r\nNeuroglian shares this domain organization and significant sequence identity with Ll, a\r\nmurine neural adhesion molecule that could be a functional homologue. Here I report the\r\ncrystal structure of a proteolytic fragment containing the first two FnIII repeats of\r\nneuroglian (NgFn 1,2) at 2.0\u00c5. The interpretation of photomicrographs of rotary\r\nshadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five\r\nneuroglian Fn III domains, is also discussed.
\r\n\r\nThe structure of NgFn 1,2 consists of two roughly cylindrical \u03b2-barrel structural motifs\r\narranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined\r\nby the cylinder axes. The folding topology of each domain is identical to that previously\r\nobserved for single FnIII domains from tenascin and fibronectin. The domains of\r\nNgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the\r\nlongest dimension of the fragment. Assuming this relative orientation is a general property\r\nof tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other\r\nproteins are modeled as thin straight rods with two domain zig-zag repeats. When\r\ncombined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4\r\nand CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 \u00c5 in\r\ndiameter) that extends up to 370\u00c5 from the cell surface.
\r\n\r\nIn photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible\r\nrod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean\r\ntotal length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 \u00c5, consistent with a head-to-tail\r\npacking of neuroglian's C2-type domains.
\r\n", "doi": "10.7907/43ng-md46", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7664", "collection": "thesis", "collection_id": "7664", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05032013-161139911", "type": "thesis", "title": "A biochemical and structural characterization of Drosophila neuroglian", "author": [ { "family_name": "Huber", "given_name": "Andrew Henry", "clpid": "Huber-Andrew-Henry" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Anderson", "given_name": "David J.", "orcid": "0000-0001-6175-3872", "clpid": "Anderson-D-J" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A number of cell-cell interactions in the nervous system are mediated by\r\nimmunoglobulin gene superfamily members. For example, neuroglian, a homophilic\r\nneural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C-\r\n2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats.\r\nNeuroglian shares this domain organization and significant sequence identity with Ll, a\r\nmurine neural adhesion molecule that could be a functional homologue. Here I report the\r\ncrystal structure of a proteolytic fragment containing the first two FnIII repeats of\r\nneuroglian (NgFn 1,2) at 2.0\u00c5. The interpretation of photomicrographs of rotary\r\nshadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five\r\nneuroglian Fn III domains, is also discussed.
\r\n\r\nThe structure of NgFn 1,2 consists of two roughly cylindrical \u03b2-barrel structural motifs\r\narranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined\r\nby the cylinder axes. The folding topology of each domain is identical to that previously\r\nobserved for single FnIII domains from tenascin and fibronectin. The domains of\r\nNgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the\r\nlongest dimension of the fragment. Assuming this relative orientation is a general property\r\nof tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other\r\nproteins are modeled as thin straight rods with two domain zig-zag repeats. When\r\ncombined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4\r\nand CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 \u00c5 in\r\ndiameter) that extends up to 370\u00c5 from the cell surface.
\r\n\r\nIn photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible\r\nrod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean\r\ntotal length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 \u00c5, consistent with a head-to-tail\r\npacking of neuroglian's C2-type domains.
\r\n", "doi": "10.7907/43ng-md46", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7631", "collection": "thesis", "collection_id": "7631", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04252013-080541344", "primary_object_url": { "basename": "Beal_pa_1994.pdf", "content": "final", "filesize": 40560455, "license": "other", "mime_type": "application/pdf", "url": "/7631/1/Beal_pa_1994.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Recognition of double helical DNA by purine oligonucleotides via triple helix formation", "author": [ { "family_name": "Beal", "given_name": "Peter A.", "orcid": "0000-0003-4855-7185", "clpid": "Beal-Peter-A" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Oligonucleotide-directed triple helix formation is one of the most\r\nversatile methods for the sequence specific recognition of double helical\r\nDNA. Chapter 2 describes affinity cleaving experiments carried out to assess\r\nthe recognition potential for purine-rich oligonucleotides via the formation\r\nof triple helices. Purine-rich oligodeoxyribonucleotides were shown to bind\r\nspecifically to purine tracts of double helical DNA in the major groove\r\nantiparallel to the purine strand of the duplex. Specificity was derived from\r\nthe formation of reverse Hoogsteen G\u2022GC, A\u2022AT and T\u2022AT triplets and\r\nbinding was limited to mostly purine tracts. This triple helical structure was\r\nstabilized by multivalent cations, destabilized by high concentrations of\r\nmonovalent cations and was insensitive to pH. A single mismatched base\r\ntriplet was shown to destabilize a 15 mer triple helix by 1.0 kcal/mole at 25\u00b0C.\r\nIn addition, stability appeared to be correlated to the number of G\u2022GC triplets\r\nformed in the triple helix. This structure provides an additional framework\r\nas a basis for the design of new sequence specific DNA binding molecules.
\r\n\r\nIn work described in Chapter 3, the triplet specificities and required\r\nstrand orientations of two classes of DNA triple helices were combined to\r\ntarget double helical sequences containing all four base pairs by alternate\r\nstrand triple helix formation. This allowed for the use of oligonucleotides\r\ncontaining only natural 3'-5' phosphodiester linkages to simultaneously bind\r\nboth strands of double helical DNA in the major groove. The stabilities and\r\nstructures of these alternate strand triple helices depended on whether the\r\nbinding site sequence was 5'-(purine)_m (pyrimidine)_n-3' or 5'-\r\n(pyrimidine)_m (purine)_n-3'.
\r\n\r\nIn Chapter 4, the ability of oligonucleotide-cerium(III) chelates to direct\r\nthe transesterfication of RNA was investigated. Procedures were developed\r\nfor the modification of DNA and RNA oligonucleotides with a hexadentate\r\nSchiff-base macrocyclic cerium(III) complex. In addition, oligoribonucleotides\r\nmodified by covalent attachment of the metal complex through two different\r\nlinker structures were prepared. The ability of these structures to direct\r\ntransesterification to specific RNA phosphodiesters was assessed by gel\r\nelectrophoresis. No reproducible cleavage of the RNA strand consistent with\r\ntransesterification could be detected in any of these experiments.
", "doi": "10.7907/5bbg-zs22", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7722", "collection": "thesis", "collection_id": "7722", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05172013-114618257", "primary_object_url": { "basename": "Ross 1994.pdf", "content": "final", "filesize": 24975103, "license": "other", "mime_type": "application/pdf", "url": "/7722/1/Ross 1994.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Insights into the Mechanism of Human Erythrocyte Hexose Transport: a Transferred NOE Study of Glucose Binding to GLUT1", "author": [ { "family_name": "Ross", "given_name": "Scott Alan", "clpid": "Ross-Scott-Alan" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Weitekamp", "given_name": "Daniel P.", "orcid": "0000-0003-0079-8000", "clpid": "Weitekamp-D-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This study examines binding of \u03b1- and \u03b2-D-glucose in their equilibrium mixture to the glucose transporter (GLUT1) in human erythrocyte membrane preparations by an ^1H NMR method, the transferred NOE (TRNOE). This method is shown theoretically and experimentally to be a sensitive probe of weak ligand-macromolecule interactions. The TRNOEs observed are shown to arise solely from glucose binding to GLUT1. Sites at both membrane faces contribute to the TRNOEs. Binding curves obtained are consistent with a homogeneous class of sugar sites, with an apparent KD which varies (from ~30 mM to ~70 mM for both anomers) depending on the membrane preparation examined. Preparations with a higher proportion of the cytoplasmic membrane face exposed to bulk solution yield higher apparent KKDs. The glucose transport inhibitor cytochalasin B essentially eliminates the TRNOE. Nonlinearity was found in the dependence on sugar concentration of the apparent inhibition constant for cytochalasin B reversal of the TRNOE observed in the \u03b1 anomer (and probably the \u03b2 anomer); such nonlinearity implies the existence of ternary complexes of sugar, inhibitor and transporter. The inhibition results furthermore imply the presence of a class of relatively high-affinity (KD < 2mM) sugar sites specific for the \u03b1 anomer which do not contribute to NMR-observable binding. The presence of two classes of sugar-sensitive cytochalasin B sites is also indicated. These results are compared with predictions of the alternating conformer model of glucose transport. Variation of apparent KD in the NMR-observable sites, the formation of ternary complexes and the presence of an anomer-specific site are shown to be inconsistent with this model. An alternate model is developed which reconciles these results with the known transport behavior of GLUT1. In this model, the transporter possesses (at minimum) three classes of sugar sites: (i) transport sites, which are alternately exposed to the cytoplasmic or the extracellular compartment, but never to both simultaneously, (ii) a class of sites (probably relatively low-affinity) which are confined to one compartment, and (iii) the high-affinity \u03b1 anomer-specific sites, which are confined to the cytoplasmic compartment.\r\n", "doi": "10.7907/nb45-1b79", "publication_date": "1994", "thesis_type": "phd", "thesis_year": "1994" }, { "id": "thesis:7395", "collection": "thesis", "collection_id": "7395", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01112013-110414331", "type": "thesis", "title": "Atomic-Scale Imaging and Spectroscopy Using Scanning Tunneling Microscopy", "author": [ { "family_name": "Youngquist", "given_name": "Michael George", "clpid": "Youngquist-Michael-George" } ], "thesis_advisor": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Kaiser", "given_name": "William J.", "clpid": "Kaiser-William-J" }, { "family_name": "Kuppermann", "given_name": "Aron", "clpid": "Kuppermann-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Advances in scanning tunneling microscopy (STM) instrumentation\r\nand applications are presented. An ultrahigh vacuum (UHV) scanning\r\ntunneling microscope incorporating computer-controlled two-dimensional\r\nsample translation and in vacuo tip and sample transfer was developed. Its\r\nperformance is documented through large-area and atomic-resolution\r\nimaging of highly stepped Si(111) 7x7 reconstructed surfaces and physisorbed\r\nclusters on graphite. An STM with automated approach and intra-Dew\u00e4r\r\nspring suspension was developed for operation in cryogenic liquids. A high\r\nperformance digital signal processor (DSP) based control system was\r\nconstructed, and software with advanced spectroscopic imaging and data\r\nprocessing capabilities was developed.
\r\n\r\nThe feasibility of individual-molecule vibrational spectroscopy via\r\nSTM-detected inelastic electron tunneling is assessed. In preliminary\r\nexperiments, a low-temperature STM was used for energy gap and phonon\r\nspectroscopy of superconducting Pb films. The first STM observation of\r\nphonon density of states effects in a superconductor is reported.
\r\n\r\nA systematic UHV STM imaging and spectroscopy study of 2H-MoS_2\r\nwas conducted. Atom-resolved images from three distinct imaging modes\r\nare presented. Occasional appearance of negative differential resistance\r\n(NOR) in I vs. V measurements is traced to changing tip electronic structure\r\nrather than localized surface states. Other potential NOR mechanisms are\r\ndiscussed including electron trap charging and resonant tunneling through a\r\ndouble-barrier quantum well structure arising from layer separation in the\r\nMoS_2 crystal.
\r\n\r\nDNA was imaged at atomic resolution with a UHV STM. Images show\r\ndouble-helical structure, base pairs, and atomic-scale substructure.\r\nExperimental STM profiles have atom-for-atom correlation with the A-DNA\r\nvan der Waals surface. This work demonstrates the potential of the STM for\r\ncharacterization of large biomolecular structures.
\r\n\r\nImpurity-pinned steps on silicon and gold surfaces were imaged by\r\nSTM. Pinned gold steps have short linear coherence lengths and form step\r\nloops at impurities by an Orowan-type bypassing mechanism. Step loops\r\nwere not observed at Si(111) pinning sites; step contours seem to be correlated\r\nwith the degree of order in the Si surface reconstruction.
", "doi": "10.7907/0d6k-be86", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7395", "collection": "thesis", "collection_id": "7395", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01112013-110414331", "type": "thesis", "title": "Atomic-Scale Imaging and Spectroscopy Using Scanning Tunneling Microscopy", "author": [ { "family_name": "Youngquist", "given_name": "Michael George", "clpid": "Youngquist-Michael-George" } ], "thesis_advisor": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "orcid": "0000-0001-8839-4822", "clpid": "Beauchamp-J-L" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Kaiser", "given_name": "William J.", "clpid": "Kaiser-William-J" }, { "family_name": "Kuppermann", "given_name": "Aron", "clpid": "Kuppermann-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Advances in scanning tunneling microscopy (STM) instrumentation\r\nand applications are presented. An ultrahigh vacuum (UHV) scanning\r\ntunneling microscope incorporating computer-controlled two-dimensional\r\nsample translation and in vacuo tip and sample transfer was developed. Its\r\nperformance is documented through large-area and atomic-resolution\r\nimaging of highly stepped Si(111) 7x7 reconstructed surfaces and physisorbed\r\nclusters on graphite. An STM with automated approach and intra-Dew\u00e4r\r\nspring suspension was developed for operation in cryogenic liquids. A high\r\nperformance digital signal processor (DSP) based control system was\r\nconstructed, and software with advanced spectroscopic imaging and data\r\nprocessing capabilities was developed.
\r\n\r\nThe feasibility of individual-molecule vibrational spectroscopy via\r\nSTM-detected inelastic electron tunneling is assessed. In preliminary\r\nexperiments, a low-temperature STM was used for energy gap and phonon\r\nspectroscopy of superconducting Pb films. The first STM observation of\r\nphonon density of states effects in a superconductor is reported.
\r\n\r\nA systematic UHV STM imaging and spectroscopy study of 2H-MoS_2\r\nwas conducted. Atom-resolved images from three distinct imaging modes\r\nare presented. Occasional appearance of negative differential resistance\r\n(NOR) in I vs. V measurements is traced to changing tip electronic structure\r\nrather than localized surface states. Other potential NOR mechanisms are\r\ndiscussed including electron trap charging and resonant tunneling through a\r\ndouble-barrier quantum well structure arising from layer separation in the\r\nMoS_2 crystal.
\r\n\r\nDNA was imaged at atomic resolution with a UHV STM. Images show\r\ndouble-helical structure, base pairs, and atomic-scale substructure.\r\nExperimental STM profiles have atom-for-atom correlation with the A-DNA\r\nvan der Waals surface. This work demonstrates the potential of the STM for\r\ncharacterization of large biomolecular structures.
\r\n\r\nImpurity-pinned steps on silicon and gold surfaces were imaged by\r\nSTM. Pinned gold steps have short linear coherence lengths and form step\r\nloops at impurities by an Orowan-type bypassing mechanism. Step loops\r\nwere not observed at Si(111) pinning sites; step contours seem to be correlated\r\nwith the degree of order in the Si surface reconstruction.
", "doi": "10.7907/0d6k-be86", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:3629", "collection": "thesis", "collection_id": "3629", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09182007-093920", "primary_object_url": { "basename": "Kim_j_1993.pdf", "content": "final", "filesize": 42564484, "license": "other", "mime_type": "application/pdf", "url": "/3629/1/Kim_j_1993.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Crystallographic structures and functional implications of nitrogenase molybdenum-iron proteins from Azotobacter vinelandii and Clostridium pasteurianum", "author": [ { "family_name": "Kim", "given_name": "Jongsun", "clpid": "Kim-J" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nThree-dimensional structures of the nitrogenase molybdenum-iron (MoFe-) proteins from Azotobacter vinelandii and Clostridium pasteurianum have been determined by X-ray crystallography. The structure of MoFe-protein from A. vinelandii (Av1) was determined at 2.7[...] by the method of multiple isomorphous replacement (MIR) and noncrystallographic symmetry (NCS) averaging both within and between crystal forms. The Av1 model has been refined to a crystallographic R factor of 19% with good geometry. The root mean square (rms) deviation of bond lengths and bond angles are 0.016[...] and 3.3\u00b0, respectively. The structure of MoFe-protein from C. pasteurianum (Cp1) was determined at 3.0[...] by a combination of molecular replacement, single isomorphous replacement (SIR) and NCS averaging both within and between crystal forms. The Cp1 model has been refined to a crystallographic R factor of 18% with good geometry. The rms deviation of and lengths and bond angles are 0.018[...] and 3.9\u00b0, respectively.\n\nThe MoFe-protein, which is an [alpha]2[beta]2 tetramer with a total molecular weight of ~240kD, contains two types of metal centers: the FeMo-cofactor and the P-cluster pair. The FeMo-cofactor is believed to represent the site of substrate reduction and the P-cluster pair may function in electron transfer between iron (Fe-) protein and the FeMo-cofactor. The FeMo-cofactor contains two clusters of composition 4Fe:3S and lMo:3Fe:3S that are bridged by three non-protein ligands. Two of the bridging ligands are assigned as sulfurs, while the chemical identity of the \"Y\" ligand is still ambiguous, but it could be sulfur. The Fe-Fe distance between bridged iron sites average ~2.5[...], suggesting that there may be some iron-iron bonding interactions, which could contribute the fourth coordination for the bridging irons. Ignoring the partial iron-iron bonding interactions between bridged irons, six of the seven Fe atoms in the FeMo-cofactor have trigonal coordination geometry, are coordinatively unsaturated, and are potential sites for N2 activation. The N2 binding site in FeMo-cofactor may be relevant to the H2 binding site in the H-cluster of Fe-hydrogenases and the O2 binding site in the Mn-center of PSII. Homocitrate, an essential component of FeMo-cofactor, is coordinated through a hydroxyl and carboxyl oxygen to the molybdenum site. The FeMo-cofactor is attached to the [alpha] subunit through two protein ligands, Cys [alpha]275 and His [alpha]442. The P-cluster pair consists of two 4Fe:4S clusters that are bridged by two cysteine thiol ligands and a disulfide bond between two of the cluster sulfurs. The P-cluster pair is attached at the interface between the [alpha] and [beta] subunits through seven protein ligands: Cys [alpha]62, Cys [alpha]88, Cys [alpha]154, Cys [beta]70, Cys [beta]95, Cys [beta]153, and Ser [beta]188. The structure of the P-cluster pair indicates that the P-cluster pair can act as a two-electron redox group, involving cleavage and reformation of the [mu]3-disulfide bridge coupled to the transfer of electrons into the FeMo-cofactor. This disulfide bond may also provide a site for H2 evolution.\n\nThe [alpha] and [beta] subunits in the [alpha]2[beta]2 MoFe-protein tetramer exhibit similar polypeptide folds consisting of three domains of [alpha]/[beta] type with some extra helices. The [alpha] and [beta] subunits of MoFe-protein are related by an approximate two-fold axis which passes through the center of the P-cluster pair, and there are two wide and shallow clefts around the P-cluster pair which may provide the binding site for the dimeric Fe-protein. Docking studies between the Fe-protein and MoFe-protein suggest a possible interaction mode between the two proteins that involves the surface of the MoFe-protein near the approximate two-fold axis passing through the P-cluster pair, and the surface of the Fe-protein near the 4Fe:4S cluster. The overall dimensions of the [alpha]2[beta]2 MoFe-protein teamer are ~70[...] x 80[...] x 110[...]. The two [alpha][beta] subunit pairs are related by a two-fold NCS axis. Even though the [alpha] and [beta] subunits in an [alpha][beta] subunit pair are also approximately related by a two-fold rotation, the MoFe-protein does not exhibit 222 symmetry. The MoFe-protein tetramer interface is stabilized by packing of helices primarily provided by two [beta] subunits, with some contribution from the [alpha] subunit, and further stabilized by divalent cation binding.\n\nThe FeMo-cofactor is buried at least 10[...] below the protein surface. No permanent channels between the protein surface and the FeMo-cofactor are present, however, there are two potential clefts which could be utilized for substrate entry/product release and/or H3O+ transport. The protein environment of the FeMo-cofactor indicates that there are multiple potential transfer pathways. The P-cluster pair is also buried about 12[...] below the protein surface and the environment of the P-cluster pair is primarily provided by hydrophobic residues. The edge-edge distance of the FeMo-cofactor to the P-cluster pair is about 14[...]. Four helices are oriented in parallel between the two metal centers and could play a role in electron transfer. In particular, the helices [alpha]63-74 and [alpha]88-92 provide the most direct structural connection between a P-cluster pair and FeMo-cofactor.\n\nThe structure of Cp1, including the two types of metal centers associated with the protein, are similar to that of Av1. Unique features of the Cp1 structure arise from the presence of a ~50 residue insertion in the [alpha] subunit and a ~50 residue deletion in the [beta] subunit. As a consequence, the FeMo-cofactor is more buried in Cp1 than in Av1, since the insertion is located on the surface above the FeMo-cofactor. The location of this insertion near the putative Fe-protein binding site provides a structural basis for the observation that the nitrogenase proteins from C. pasteurianum have low activity with complementary nitrogenase proteins isolated from other organisms.\n", "doi": "10.7907/bnbs-6507", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:5318", "collection": "thesis", "collection_id": "5318", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10212009-152426877", "primary_object_url": { "basename": "Evans_js_1993.pdf", "content": "final", "filesize": 17040139, "license": "other", "mime_type": "application/pdf", "url": "/5318/1/Evans_js_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "NMR and Computational Studies on the Conformational Folding of the Biomineralization Template, Posphophoryn", "author": [ { "family_name": "Evans", "given_name": "John Spencer", "orcid": "0000-0002-9565-7296", "clpid": "Evans-John-Spencer" } ], "thesis_advisor": [ { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Bovine Dentine Phosphophoryn (BDPP) is a member of the \"Asp-rich\" superfamily of template macromolecules known as polyelectrolyte mineral matrix proteins (PMMPs). Using 1-D and 2-D NMR multinuclear spectroscopy, protein sequencing, solid-phase peptide synthesis, and molecular modeling, we investigated the capacity of BDPP to fold in solution under certain conditions. It is believed that the folding properties of a template macromolcule are important in the formation of n inorganic mineral phase. In this report, we have established the following regarding BDPP structure:\r\n\r\nI. BDPP sequence organization can be conceptualized as three types of domains: polyelectrolyte calcium binding domains (PCBD), hinge \r\ndomains (HD), and the hydrophobic domains (HC). The PCBD regions can be further subdivided into (Asp)_n, (PSer)_n, and (PSerAsp)_n-containing\r\nhomopolymer and heteropolymer sequence stretches. The HD contain predominantly neutral or uncharged amino acids such as Ser, Gly, and \r\nPro. These HD domain sequences are flanked on either side by PCBD regions.\r\n\r\nII. BDPP contains a number of Lys sidechains (44/1000 residues) which are believed to form ion pairs with either Asp, Glu, or PSer residues \r\nin the protein. The function of this salt-bridging is not understood at this time, but it may be responsible for maintaining the protein template molecule in a conformation that retains a high negative charge density.\r\n\r\nIII. At low pH, or in the presence of divalent cations, BDPP assumes a global conformation that is condensed in particle size. At neutral pH \r\nunder conditions of low ionic strength and in the absence of divalent cations, this global conformation converts to an extended form. The \r\nfolding transition between these two conformers is mediated by conformational change in the BD regions which are flanked by specific \r\nPCBD sequence regions.\r\n\r\nIV. The PCBD regions possess some degree of tertiary and secondary structural organization in the absence of divalent cations at low ionic \r\nstrength. This folding permits the surface charge density of these regions to remain high, relative to that of a random coil conformer.\r\n\r\nV. BDPP exhibits a selectivity in terms of divalent cation binding sites. Under conditions of low ionic strength and divalent cation depletion, the addition of Cd (II) to BDPP leads to binding at various PCBD sequence stretches, according to the following order:\r\n(PSer)_n > (PserAsp)_n > (Asp)_n\r\n\r\nVI. Modeling studies conducted on PCBD sequence peptides [(Asp)_(20), (PSer)_(20), and (PSerAsp)_(10)] indicate that there is a sequence preference for certain conformers in the presence of Na^+, i.e., (Asp)_(20) forms \"supercoils\", (PSer)_(20) forms \"hairpins\", and (PSerAsp)_(10) forms \"spirals\" or \"distorted\" hairpins. Each of these conformers features some degree of sidechain folding and/or peptide backbone secondary structure, in support of Mann's hypothesis, as well as the experimental data obtained for BDPP.\r\n\r\nVII. A peptide mimetic which represents a PCBD-Hinge-PCBD motif of rat \u03b1-phosphophoryn, was constructed using FMOC solid-phase peptide \r\nsynthesis. Using 2-D NMR spectroscopy and monitoring \u03b1-CH and sidechain \u03b2-CH_2 proton chemical shifts, we can demonstrate that this \r\npeptide mimetic folds or collapses under conditions of low pH and divalent cation addition, in a manner similar to that observed for intact \r\nBDPP.\r\n", "doi": "10.7907/ky1z-w552", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7275", "collection": "thesis", "collection_id": "7275", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11192012-103302776", "primary_object_url": { "basename": "Clark_sm_1993.pdf", "content": "final", "filesize": 37671547, "license": "other", "mime_type": "application/pdf", "url": "/7275/1/Clark_sm_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Advances in scanning force microscopy of biological structures", "author": [ { "family_name": "Clark", "given_name": "Steven Manning", "clpid": "Clark-Steven-Manning" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A multifacted approach to the imaging of biological structures by scanning force microscopy is described. The major problems addressed are the distortion of biological samples by excessive forces applied by the cantilever stylus and sample motion relative to the imaging substrate.
\r\n\r\nThe first two chapters discuss the design of digital signal processor based scanning force microscope control electronics and a novel microscope head that eliminates the application of excessive forces to the sample caused\r\nby electronic or vibrational noise.
\r\n\r\nThe third chapter presents a novel use of chemical vapor deposition for application of heterofunctional alkoxysilanes to scanning force microscopy imaging subsrates. This technique provides imaging substrates which have chemical groups that can be used for sample immobilization without compromising substrate smoothness. The use of the chemically derivatized substrates for scanning force microscopy is also explored.
\r\n\r\nThe final chapter presents high resolution images of bovine liver catalase micro-crystals. The images of the protein micro-crystals show resolution on the order of 2 to 3 nanometers allowing the visualization of individual catalase tetramers. To our knowledge this is the first report of images of protein micro-crystals taken by scanning force microscopy which have resolution comparable to that of electron microscopy.
\r\n", "doi": "10.7907/0vhp-pk35", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:7275", "collection": "thesis", "collection_id": "7275", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11192012-103302776", "primary_object_url": { "basename": "Clark_sm_1993.pdf", "content": "final", "filesize": 37671547, "license": "other", "mime_type": "application/pdf", "url": "/7275/1/Clark_sm_1993.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Advances in scanning force microscopy of biological structures", "author": [ { "family_name": "Clark", "given_name": "Steven Manning", "clpid": "Clark-Steven-Manning" } ], "thesis_advisor": [ { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Revel", "given_name": "Jean-Paul", "clpid": "Revel-J-P" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Fraser", "given_name": "Scott E.", "orcid": "0000-0002-5377-0223", "clpid": "Fraser-S-E" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Wold", "given_name": "Barbara J.", "orcid": "0000-0003-3235-8130", "clpid": "Wold-B-J" }, { "family_name": "Hood", "given_name": "Leroy E.", "orcid": "0000-0001-7158-3678", "clpid": "Hood-L-E" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "A multifacted approach to the imaging of biological structures by scanning force microscopy is described. The major problems addressed are the distortion of biological samples by excessive forces applied by the cantilever stylus and sample motion relative to the imaging substrate.
\r\n\r\nThe first two chapters discuss the design of digital signal processor based scanning force microscope control electronics and a novel microscope head that eliminates the application of excessive forces to the sample caused\r\nby electronic or vibrational noise.
\r\n\r\nThe third chapter presents a novel use of chemical vapor deposition for application of heterofunctional alkoxysilanes to scanning force microscopy imaging subsrates. This technique provides imaging substrates which have chemical groups that can be used for sample immobilization without compromising substrate smoothness. The use of the chemically derivatized substrates for scanning force microscopy is also explored.
\r\n\r\nThe final chapter presents high resolution images of bovine liver catalase micro-crystals. The images of the protein micro-crystals show resolution on the order of 2 to 3 nanometers allowing the visualization of individual catalase tetramers. To our knowledge this is the first report of images of protein micro-crystals taken by scanning force microscopy which have resolution comparable to that of electron microscopy.
\r\n", "doi": "10.7907/0vhp-pk35", "publication_date": "1993", "thesis_type": "phd", "thesis_year": "1993" }, { "id": "thesis:6671", "collection": "thesis", "collection_id": "6671", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09152011-080428062", "primary_object_url": { "basename": "Wang_w_1992.pdf", "content": "final", "filesize": 56468825, "license": "other", "mime_type": "application/pdf", "url": "/6671/1/Wang_w_1992.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Expression, structural and functional studies of fasciclin I", "author": [ { "family_name": "Wang", "given_name": "Wen-Ching", "clpid": "Wang-Wen-Ching" } ], "thesis_advisor": [ { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "orcid": "0000-0002-2277-3990", "clpid": "Bjorkman-P-J" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Davidson", "given_name": "Norman R.", "clpid": "Davidson-N-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Fasciclin I is a cell surface glycoprotein thought to be involved in growth cone guidance in the embryonic insect nervous system. It is expressed on the cell surfaces of all\r\nperipheral nervous system (PNS) axons, a subset of central nervous system (CNS) axons and on some nonneuronal cells. Fly embryos bearing mutations eliminating expression of\r\nboth fasciclin I and the Abelson tyrosine kinase exhibit a severe phenotype in which many axon pathways fail to form. Fasciclin I mediates homophilic adhesion in transfected tissue culture cells, suggesting that it may affect growth cone guidance through homophilic interactions. To facilitate structure-function studies of fasciclin I, we have generated mammalian (CHO) cell lines expressing fasciclin I at a high level. The expressed fasciclin I\r\nprotein was released from the cell surface in a soluble form by phospholipase C treatment. Milligram quantities of soluble expressed fasciclin I were purified on an immunoaffinity column. Large single crystals were obtained that diffracted to ~5 \u00c5 resolution which is\r\ninsufficient for a structure determination to atomic resolution by x-ray crystallography. In an effort to produce a form of fasciclin I more amenable to crystallization, we also generated CHO and Drosophila cell (S2) lines that produce a truncated form of fasciclin I.\r\nThe soluble fasciclin I expressed in S2 cells contains significantly less carbohydrate (~15 kDa) as compared to the molecules expressed in CHO cells. Therefore, S2-derived\r\nfasciclin I may be more suitable for crystallization. Biochemical characterization of the expressed fasciclin I indicates that fasciclin I exists as a monomer in solution, an observation consistent with homophilic interaction properties only if the interaction is of low affinity. Electron micrographs of fasciclin I suggest that it has a compact rectangular shape with no obvious flexible linker regions, in contrast to what has been seen in electron\r\nmicroscopic studies of other adhesion molecules. Circular dichroism analysis suggests that fasciclin I contains significant amounts of \u03b1-helical structure, which together with the electron microscopic results, suggests that its structure is substantially different from the \u03b2-sheet structures predicted for adhesion molecules that are members of the immunoglobulin superfamily and/or contain fibronectin III repeats. Future structural and functional studies of fasciclin I will ultimately increase our understanding of neuronal cell surface recognition\r\nand axon guidance.\r\n", "doi": "10.7907/ykbt-jt41", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:6668", "collection": "thesis", "collection_id": "6668", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09132011-160134197", "primary_object_url": { "basename": "Sun_y_1992.pdf", "content": "final", "filesize": 60630395, "license": "other", "mime_type": "application/pdf", "url": "/6668/1/Sun_y_1992.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "NMR Studies of Protein-DNA Interactions: Determinations of DNA Structures Recognized by Bin Recombinase and Studies of Their Roles in Protein Binding Interactions", "author": [ { "family_name": "Sun", "given_name": "Yun", "clpid": "Sun-Yun" } ], "thesis_advisor": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "thesis_committee": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The solution structures of two DNA oligomers were determined by the 2-D NMR method. These 14-base-pair DNA molecules contain the recognition sites for Hin recombinase. In spite of the differences in their sequences, the two structures are remarkably similar. The refined DNA structures possess a significant bend (25-32\u00b0) in the middle of the helices. As a result of the bending, the nearby major groove is compressed at almost exactly the position where the recombinase binds. The DNA molecules were also found to have a deepened and narrowed minor groove near the continuous dA tracts, where the minor groove contact happens between the N-terminal residues of the recombinase binding domain and the DNA molecules. Such pre-existing unique features of the free DNA molecules are likely to contribute to the specific interaction of the protein and the DNA tracts. Structure determinations by the NMR method were preceded with the use of complete relaxation matrix analysis and restrained molecular dynamics. A data processing system were developed which allowed us to simulate the NMR spectra and quantifying intensities from an overlapped data set. A complete system for high-resolution structure determinations in solution were set up and evaluated.
\r\n\r\nThe conformation of the Hin 52mer peptide-the binding domain of the Hin recombinase-and its binding interactions with the DNA oligomers are studied by NMR, circular dichroism and chromatographic methods. The conclusion is that the peptide does not have a unique and stable conformation alone as a single monomer in solution. The Hin peptide can be prevented from being aggregated by adjusting to acidic conditions, and it can be folded to a stable tertiary structure in an artificial environment with small amounts of trifluoroethanol. The Hin 52mer peptide conformation is greatly stabilized or induced by the presence of the DNA bearing specific binding sequences. The DNA binding activities of the peptide may be assayed by a chromatographic method. The behavior of the peptide in the binding complex and the characteristic structural features of the DNA molecules suggest the active role of the DNA in protein-DNA interactions providing complementary interactions with the peptide and stabilizing the peptide conformation upon its binding.
\r\n", "doi": "10.7907/em1y-p761", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:3136", "collection": "thesis", "collection_id": "3136", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08152007-074128", "primary_object_url": { "basename": "Steele_cs_1992.pdf", "content": "final", "filesize": 8115425, "license": "other", "mime_type": "application/pdf", "url": "/3136/1/Steele_cs_1992.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Affinity : a Concurrent Programming System for Multicomputers", "author": [ { "family_name": "Steele", "given_name": "Craig Stanley", "clpid": "Steele-Craig-Stanley" } ], "thesis_advisor": [ { "family_name": "Seitz", "given_name": "Charles L.", "clpid": "Seitz-C-L" } ], "thesis_committee": [ { "family_name": "Seitz", "given_name": "Charles L.", "clpid": "Seitz-C-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Chandy", "given_name": "K. Mani", "clpid": "Chandy-K-M" }, { "family_name": "Abu-Mostafa", "given_name": "Yaser S.", "clpid": "Abu-Mostafa-Y-S" }, { "family_name": "Taylor", "given_name": "Stephen", "clpid": "Taylor-S" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "Affinity is an experiment to explore a simple, convenient, and expressive programming model that provides adequate power for complex programming tasks while setting few constraints on potential concurrency. Although the programmer is required to formulate a computational problem explicitly into medium-sized pieces of data and code, most of the additional functions necessary for concurrent execution are implicit. The execution of the light-weight, reactive processes, called actions, implicitly induces atomicity and consistency of data modifications. The programmer accesses shared data structures in a shared-memory fashion, but without the need for explicit locking to manage the problems of concurrent access and mutual exclusion. Program control flow is distributed and implicit.\r\n\r\nThe name given to the programming model, Affinity, has a definition, \"causal connection or relationship,\" that is fitting to the way programs are structured and scheduled.\r\n\r\nAffinity consistency and coherence properties provide a tractable discipline for the dangerous power of a concurrent, shared-memory programming style. Existing programming complexity-management techniques such as object-oriented languages can be used in this multicomputer environment. Affinity programs can compute consistent and correct results despite staleness of data, and asynchrony and nondeterminism in execution of code. Program correctness is invariant under replication, or cloning, of actions. This aspect of the model yields a simple and robust mechanism for fault-tolerance.\r\n\r\nThe practicality of the Affinity programming model has been demonstrated by an implementation on a second-generation multicomputer, the Ametek S/2010. The implementation is distributed, scalable, and relatively insensitive to network latency. Affinity has demonstrated reasonable efficiency and performance for computations with tens of processing nodes, hundreds of actions, and thousands of shared data structures.", "doi": "10.7907/syrm-sx30", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:6668", "collection": "thesis", "collection_id": "6668", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09132011-160134197", "primary_object_url": { "basename": "Sun_y_1992.pdf", "content": "final", "filesize": 60630395, "license": "other", "mime_type": "application/pdf", "url": "/6668/1/Sun_y_1992.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "NMR Studies of Protein-DNA Interactions: Determinations of DNA Structures Recognized by Bin Recombinase and Studies of Their Roles in Protein Binding Interactions", "author": [ { "family_name": "Sun", "given_name": "Yun", "clpid": "Sun-Yun" } ], "thesis_advisor": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "thesis_committee": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The solution structures of two DNA oligomers were determined by the 2-D NMR method. These 14-base-pair DNA molecules contain the recognition sites for Hin recombinase. In spite of the differences in their sequences, the two structures are remarkably similar. The refined DNA structures possess a significant bend (25-32\u00b0) in the middle of the helices. As a result of the bending, the nearby major groove is compressed at almost exactly the position where the recombinase binds. The DNA molecules were also found to have a deepened and narrowed minor groove near the continuous dA tracts, where the minor groove contact happens between the N-terminal residues of the recombinase binding domain and the DNA molecules. Such pre-existing unique features of the free DNA molecules are likely to contribute to the specific interaction of the protein and the DNA tracts. Structure determinations by the NMR method were preceded with the use of complete relaxation matrix analysis and restrained molecular dynamics. A data processing system were developed which allowed us to simulate the NMR spectra and quantifying intensities from an overlapped data set. A complete system for high-resolution structure determinations in solution were set up and evaluated.
\r\n\r\nThe conformation of the Hin 52mer peptide-the binding domain of the Hin recombinase-and its binding interactions with the DNA oligomers are studied by NMR, circular dichroism and chromatographic methods. The conclusion is that the peptide does not have a unique and stable conformation alone as a single monomer in solution. The Hin peptide can be prevented from being aggregated by adjusting to acidic conditions, and it can be folded to a stable tertiary structure in an artificial environment with small amounts of trifluoroethanol. The Hin 52mer peptide conformation is greatly stabilized or induced by the presence of the DNA bearing specific binding sequences. The DNA binding activities of the peptide may be assayed by a chromatographic method. The behavior of the peptide in the binding complex and the characteristic structural features of the DNA molecules suggest the active role of the DNA in protein-DNA interactions providing complementary interactions with the peptide and stabilizing the peptide conformation upon its binding.
\r\n", "doi": "10.7907/em1y-p761", "publication_date": "1992", "thesis_type": "phd", "thesis_year": "1992" }, { "id": "thesis:4683", "collection": "thesis", "collection_id": "4683", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-11292005-133156", "primary_object_url": { "basename": "Koh_js_1991.pdf", "content": "final", "filesize": 6742098, "license": "other", "mime_type": "application/pdf", "url": "/4683/1/Koh_js_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Design of novel bases for recognition of GC base pairs by oligonucleotide-directed triple helix formation", "author": [ { "family_name": "Koh", "given_name": "Jong Sung", "clpid": "Koh-J-S" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Imperiali", "given_name": "Barbara", "clpid": "Imperiali-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nPart I: Triple Helix Formation by Oligonucleotide Analogs on Double-Stranded DNA\n\nChapter 1: Design of Novel Bases for pH-Independent Recognition of GC Base Pairs by Triple Helix Formation\n\nThe ability to design synthetic molecules that bind sequence-specifically to unique sites on human DNA could have major applications in the treatment of genetic, neoplastic, and viral diseases. One powerful approach to sequence-specific binding of double helical DNA is oligonucleotide-directed triple helix formation. Specificity arises from the base triplets ([...] and C+GC) formed by Hoogsteen base pairing of the second pyrimidine strand with the purine strand of the double helix. Because protonation of the N3 is required for cytosine, triple helix formation at rich sequences is limited to a narrow pH range. The novel base 3-methyl-5-amino-7H-pyrazolo(4, 3-d)pyrimidine-7-one (P1) specifically recognizes GC base pairs as selectively and strongly as C+, but with greater affinity and over an extended pH range. Such selectivities allow binding at a 15 base pair site in pDMAG10 DNA (pH 7.8) containing [...] and at a 16 base pair site in pHIV-CAT DNA (pH 7.4, 37[degrees]C) containing [...].\n\nChapter 2: Extension of Triple Helix Formation. Design of Novel Bases for Recognition of CG Base Pairs\n\nOligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double-helical DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts containing AT and GC base pairs, it is desirable to study whether oligonucleotides can be designed to bind to all four base pairs. A general solution would allow targeting of oligonucleotides to any sequence. The novel base 3-methyl-5-amino-7H-pyrazolo(4, 3-d)pyrimidine-7-one (P1) and cytosine (C) moderately recognize CG base pairs. Such specificities allow binding at an 18 base pair site in SV 40 (pH 7.0, 37[degrees]C) DNA containing all four base pairs.\n\nPart II: Design of DNA Cleaving Groups\n\nChapter 3: Design of New DNA Cleaving Fuctional Groups and Studies of Their DNA Cleaving Mechanisms\n\nThe utility of the DNA cleaving molecules is enormous, ranging from the creation of synthetic restriction enzymes for use by molecular biologists to the development of chemotherapeutic agents which may be effective against a variety of neoplastic diseases. We synthesized three compounds: 12 (P3-Ga-His), 19 (P3-Ga-PYML), and 23 (P3-Ga-Phe). Compound 12 shows sequence-specific cleavage in the presence of Cu(II) and dioxygen, while compound 19 shows sequence-specific cleavage in the presence of dithiothreitol and dioxygen. Interestingly, compound 23 shows strong cleavage at a single site in 167 base pair fragment (EcoRI/RsaI) from plasmid pBR322 in the presence of UV light and [beta]-carbonato(trien)cobalt(III)perchlorate complex. The end product analysis of the cleaved oligonucleotide shows 5'-phosphate, 3'-phosphate, and an unknown 3'-product.\n", "doi": "10.7907/9655-mf61", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" }, { "id": "thesis:17527", "collection": "thesis", "collection_id": "17527", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07152025-155435040", "primary_object_url": { "basename": "Rivkin_LZ_1986.pdf", "content": "final", "filesize": 31977647, "license": "other", "mime_type": "application/pdf", "url": "/17527/1/Rivkin_LZ_1986.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Damping Ring for the SLAC Linear Collider", "author": [ { "family_name": "Rivkin", "given_name": "Leonid Zinovy", "orcid": "0000-0003-1671-4844", "clpid": "Rivkin-Leonid-Zinovy" } ], "thesis_advisor": [ { "family_name": "Barish", "given_name": "Barry C.", "orcid": "0000-0001-6386-7371", "clpid": "Barish-B-C" } ], "thesis_committee": [ { "family_name": "Barish", "given_name": "Barry C.", "orcid": "0000-0001-6386-7371", "clpid": "Barish-B-C" }, { "family_name": "Peck", "given_name": "Charles W.", "clpid": "Peck-C-W" }, { "family_name": "Barnes", "given_name": "Charles A.", "clpid": "Barnes-C-A" }, { "family_name": "Politzer", "given_name": "Hugh David", "orcid": "0000-0002-4983-6621", "clpid": "Politzer-H-D" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The results of a comprehensive set of measurements on the first purposeful 1.21\r\nGe V high tune, high field damping ring for the SLAC Linear Collider are reported.
\r\n\r\nCurrent dependent effects such as parasitic mode losses, head tail instabilities,\r\nsynchrotron and betatron frequency shifts were measured to estimate the\r\nimpedance. All results agree reasonably well with expectations and indicate no\r\nlimitations to the design performance. A current of 55 mA (4 \u00b7 1010 particles),\r\nwhich represents 80% of the design intensity, has been successfully stored in the\r\nring with no sign of instabilities.
\r\n\r\nSome changes to the optics design that are being incorporated into the positron\r\nand electron damping rings now under construction are described. They are based\r\non the operating experience with the present electron damping ring and include\r\nan improvement of the bending magnets and strengthening of the chromaticity\r\ncorrection scheme. As a consequence, the normalized equilibrium transverse\r\nemittance of the beam is expected to be 16 \u03bc mrad.
\r\n\r\nSome properties of possible lattices for a future very high energy linear collider\r\ndamping ring are discussed in the appendices.
", "doi": "10.7907/bgaj-xg03", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" }, { "id": "thesis:17527", "collection": "thesis", "collection_id": "17527", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07152025-155435040", "primary_object_url": { "basename": "Rivkin_LZ_1986.pdf", "content": "final", "filesize": 31977647, "license": "other", "mime_type": "application/pdf", "url": "/17527/1/Rivkin_LZ_1986.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Damping Ring for the SLAC Linear Collider", "author": [ { "family_name": "Rivkin", "given_name": "Leonid Zinovy", "orcid": "0000-0003-1671-4844", "clpid": "Rivkin-Leonid-Zinovy" } ], "thesis_advisor": [ { "family_name": "Barish", "given_name": "Barry C.", "orcid": "0000-0001-6386-7371", "clpid": "Barish-B-C" } ], "thesis_committee": [ { "family_name": "Barish", "given_name": "Barry C.", "orcid": "0000-0001-6386-7371", "clpid": "Barish-B-C" }, { "family_name": "Peck", "given_name": "Charles W.", "clpid": "Peck-C-W" }, { "family_name": "Barnes", "given_name": "Charles A.", "clpid": "Barnes-C-A" }, { "family_name": "Politzer", "given_name": "Hugh David", "orcid": "0000-0002-4983-6621", "clpid": "Politzer-H-D" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_pma" } ], "abstract": "The results of a comprehensive set of measurements on the first purposeful 1.21\r\nGe V high tune, high field damping ring for the SLAC Linear Collider are reported.
\r\n\r\nCurrent dependent effects such as parasitic mode losses, head tail instabilities,\r\nsynchrotron and betatron frequency shifts were measured to estimate the\r\nimpedance. All results agree reasonably well with expectations and indicate no\r\nlimitations to the design performance. A current of 55 mA (4 \u00b7 1010 particles),\r\nwhich represents 80% of the design intensity, has been successfully stored in the\r\nring with no sign of instabilities.
\r\n\r\nSome changes to the optics design that are being incorporated into the positron\r\nand electron damping rings now under construction are described. They are based\r\non the operating experience with the present electron damping ring and include\r\nan improvement of the bending magnets and strengthening of the chromaticity\r\ncorrection scheme. As a consequence, the normalized equilibrium transverse\r\nemittance of the beam is expected to be 16 \u03bc mrad.
\r\n\r\nSome properties of possible lattices for a future very high energy linear collider\r\ndamping ring are discussed in the appendices.
", "doi": "10.7907/bgaj-xg03", "publication_date": "1986", "thesis_type": "phd", "thesis_year": "1986" } ]