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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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: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:10783", "collection": "thesis", "collection_id": "10783", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03302018-093456214", "primary_object_url": { "basename": "Kielkopf_CL_2000.pdf", "content": "final", "filesize": 46362175, "license": "other", "mime_type": "application/pdf", "url": "/10783/1/Kielkopf_CL_2000.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structural Basis of DNA Recognition by Synthetic Ligands", "author": [ { "family_name": "Kielkopf", "given_name": "Clara Louise", "clpid": "Kielkopf-Clara-Louise" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The DNA double helix presents functional groups in the major and minor grooves that can be used by ligands for readout of the base pair sequence. The overall flexibility and shape also distinguishes various sequences. Although the information displayed in the major groove is more diverse, small DNA-binding polyamides predictably distinguish all four base pairs in the minor groove. In this work, the structural basis of this recognition has been studied using x-ray crystallographic techniques, and is described for G\u2022C (Chapter 2) and T\u2022A base pairs (Chapter 3-4). The polyamides directly read the DNA sequence using a combination of specific hydrogen bonds and shape selection. A second class of ligands called intercalators bind to DNA via the major groove by slipping a planar aromatic ligand between the bases. Although this shape-selective DNA binding is relatively nonspecific, the substitution of the ancillary ligands on octahedral metallointercalators allows specific sequences to be recognized in the major groove. The high resolution crystal structure of a designed metal complex bound to its target site reveals the nature of these specific contacts, as well as the precise stacking of the ligand between the bases. A detailed understanding of specific DNA recognition by small molecules is important for their further development as tools for molecular biology and medicine. These structures reveal how synthetic ligands can distinguish all four base pairs in both the major and the minor grooves of DNA.", "doi": "10.7907/73K1-K290", "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:10463", "collection": "thesis", "collection_id": "10463", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09272017-143047601", "primary_object_url": { "basename": "Schlessman_jl_1997.pdf", "content": "final", "filesize": 31028787, "license": "other", "mime_type": "application/pdf", "url": "/10463/1/Schlessman_jl_1997.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Crystal Structure of Azotobachter vinelandii Nitrogenase Iron Protein at 2.2 A\u030a Resolution", "author": [ { "family_name": "Schlessman", "given_name": "Jamie L.", "clpid": "Schlessman-Jamie-L" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Biological nitrogen fixation by the two-component metalloenzyme nitrogenase \r\nprovides an elegant solution to the problem of reducing abundant, but relatively inert, \r\ndinitrogen to the biologically usable ammonia needed by all organisms. This oxygen \r\nsensitive enzyme, consisting of the separately purifiable nitrogenase iron protein and \r\nmolybdenum iron protein, couples nucleotide hydrolysis to electron transfer to catalyze the \r\nATP-dependent reaction. Iron protein acts as the sole known biological reductant to \r\nmolybdenum iron protein, which contains the actual site of substrate reduction. MgATP \r\nbinding to iron protein induces dramatic conformational changes in the protein's structure \r\nrequired for docking with molybdenum iron protein. Complex formation and dissociation \r\nare essential for nucleotide hydrolysis, electron transfer, and substrate reduction.
\r\n\r\n\r\nWe have determined the crystal structure of Azotobacter vinelandii nitrogenase iron \r\nprotein at 2.2 A\u030a resolution in the absence of nucleotide. Crystals grew in space group\r\nP212121, and represent a new crystal form compared with that of the structure previously determined at 2.9 A\u030a resolution. X-ray diffraction data were collected from a single crystal\r\nusing cryocrystallographic techniques. The structure was solved by molecular replacement, \r\nfollowed by solvent flattening and noncrystallographic averaging. The current model \r\ncontains 575 of 578 possible amino acid residues and 372 solvent molecules, and has been \r\nrefined to R-value of 22.3 % (R-free = 29.0 %) for all data to 2.2 A\u030a, with good stereochemistry.
\r\n\r\n\r\nThe overall topology of nitrogenase iron protein consists of an Fe4S4 cluster symmetrically ligated by two identical subunits of doubly wound \u03b1/\u03b2 structure similar to\r\nthose of other nucleotide binding proteins. A detailed description is provided of those structural features important for iron protein function, including nucleotide binding regions, the Fe4S4 cluster environment, intersubunit interactions, and the molybdenum iron protein\r\nbinding surface. Comparisons are made between the current model and that of C.\r\npasteurianum iron protein, as well as those of two A. vinelandii nitrogenase complexes. \r\nAnalysis of the various iron protein structures provides a framework for considering the \r\nwealth of relevant nitrogenase spectroscopic, biochemical, and genetic information.
\r\n \r\n\r\n", "doi": "10.7907/8bav-4095", "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: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: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:7392", "collection": "thesis", "collection_id": "7392", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01102013-134402009", "type": "thesis", "title": "Structure-function studies of fibroblast growth factors (FGFS)", "author": [ { "family_name": "Zhu", "given_name": "Xiaotian", "clpid": "Zhu-Xiaot" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Unknown", "given_name": "Unknown" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The fibroblast growth factor (FGF) family exhibits mitogenic, chemotactic and\r\nangiogenic activity in a variety of cell types. The first three-dimensional structures of\r\ntwo members of the FGF family, bovine acidic FGF (aFGF) and human basic FGF\r\n(bFGF), have been crystallographically determined by multiple isomorphous\r\nreplacement (MIR), and refined to 2.7 \u00c5 and 1.9 \u00c5 respectively. The structures of\r\nboth aFGF and bFGF consist of twelve antiparallel \u03b2 strands which are arranged in a\r\nfolding pattern with approximate three-fold internal symmetry. A striking feature of\r\nthe FGF structures is the overall similarity to the structures of soybean trypsin\r\ninhibitor and interleukins-1\u03b1 and 1\u03b2, in spite of the low sequence homology between\r\nthese proteins.
\r\n\r\nFGF stimulates cellular proliferation and differentiation through the interactions\r\nwith both the cell surface FGF receptor and heparin. In the FGF structures, the two\r\nputative receptor binding sites are located on different sides of FGF. Also, a region\r\nrich in positively charged amino acids that is likely involved in heparin binding has\r\nbeen found in the FGF structures. It is further shown that the putative heparin and\r\nreceptor binding regions occupy distinct locations on the protein surface.
\r\n\r\nBecause heparin is required for FGF binding to its receptor, the interactions\r\nbetween FGF and sucrose octasulfate, a heparin analog, have been studied. The\r\ncrystal structure of the complex between aFGF and sucrose octasulfate has been\r\ndetermined to 2.7 \u00c5 resolution by a combination of MIR and molecular replacement\r\nmethods. Sucrose octasulfate binds to the aFGF positive patch mentioned above as a\r\npotential heparin binding site. Based on the structure of aFGF and sucrose octasulfate complex, a possible FGF receptor binding mechanism in the presence of heparin is\r\nproposed.
\r\n\r\nOther crystallographic studies of FGF include the structural determination of the\r\ntwo FGF mutants; the complex of aFGF and 1,3,6-naphthalene trisulfonate, a close\r\nanalog of the FGF inhibitor suramin; and the bFGF-copper complex.
\r\n", "doi": "10.7907/d0f2-e676", "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" } ]