@conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101402, title ="Engineering mechanical properties in thin films of bacterial cells", author = "Sim, Seunghyun and Hui, Yue", pages = "POLY-0085", month = "March", year = "2020", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200219-152527339", note = "© 2020 American Chemical Society.", revision_no = "9", abstract = "This lecture will describe our ongoing effort to control the mech. properties of thin bacterial films by display of adhesive proteins on the cell surface and by release of matrix proteins into the extracellular space. Studies of film fabrication, cell viability, film growth, film structure, indentation behavior, and regeneration following injury will be discussed.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101397, title ="Genetically encoded 3,4-ethylenedioxythiophene (EDOT) functionality for fabrication of protein-based conductive polymers", author = "Obana, Maiko and Tirrell, David Anthony", pages = "BIOT-0049", month = "March", year = "2020", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200219-141928447", note = "© 2020 American Chemical Society.", revision_no = "9", abstract = "Conductive polymers are an important class of materials because of their unique electronic and optical properties. Unlike inorg. conductors, conductive polymers are easily functionalized and processed, and therefore, are widely utilized at the interface with biol. (e.g. biosensors, cell culture, neural probes, drug delivery). By linking conductive polymers and proteins via genetically incorporated monomer units, we are developing protein-based conductive materials. Because the size and sequence of proteins are precisely controlled by gene expression, we can fine tune the design of the materials such as the no. of monomer units in a protein and the chem. environment around the monomer. In the presentation, we will demonstrate genetic incorporation of a non-canonical amino acid (designated EDOT-Lys) bearing the 3,4-ethylenedioxythiophene (EDOT) group, which is subsequently polymd. to form conductive polymers. E. coli expressing mutants of M. mazei pyrrolysyl-tRNA synthetase (mmPylRS) revealed strong fluorescence from GFP as an indication of successful incorporation of EDOT-Lys. We will also present polymn. of the unstructured protein XTEN and small peptides through their pendant EDOT groups.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101379, title ="Mapping chain unfolding in macromolecular networks under stress", author = "Baccile, Joshua and Tirrell, David Anthony", pages = "POLY-0541", month = "March", year = "2020", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200219-113331019", note = "© 2020 American Chemical Society.", revision_no = "9", abstract = "We have prepd. well-defined macromol. networks that contain engineered fibronectin domains that unfold when subjected to mech. stress. Unfolding of the network chains can be monitored by observing changes in Forster resonance energy transfer (FRET) between pairs of dyes that flank the fibronectin domains. This lecture will describe the spatial mapping of FRET efficiency in protein hydrogels subject to non-uniform mech. stress fields.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101365, title ="Selective proteomic analysis of cellular sub-populations in complex biological systems", author = "Tirrell, David Anthony", pages = "MPPG-0013", month = "March", year = "2020", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200219-084532646", note = "© 2020 American Chemical Society.", revision_no = "9", abstract = "This lecture will describe the use of non-canonical amino acids (ncAAs) as selective probes of protein synthesis in complex biol. systems. Pulse-labeling with ncAA probes provides time-resoln., while controlled expression of mutant aminoacyl-tRNA synthetases allows the investigator to restrict anal. to cell types or cell states of interest. The methods are applicable to studies of microbial systems, mammalian cell culture, and a variety of animal models. The scope and limitations of the approach, and some of our most recent results, will be discussed.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/101199, title ="The Effect of Cryo Temperature on Commonly used Fluorophores", author = "Metskas, Lauren Ann and Ho, Samuel", journal = "Biophysical Journal", volume = "118", number = "3", pages = "150a-151a", month = "February", year = "2020", doi = "10.1016/j.bpj.2019.11.942", issn = "0006-3495", url = "https://resolver.caltech.edu/CaltechAUTHORS:20200210-110813112", note = "© 2020 Biophysical Society. \n\nAvailable online 7 February 2020.", revision_no = "10", abstract = "Correlated cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) has become an increasingly popular method for combining the resolving power of cryo-EM with the specificity of fluorescence. Although cryo-fluorescence microscopy suffers from optical limitations, it is a powerful way to target the resolving power of cryo-EM toward proteins of interest in heterogeneous cellular environments. Super-resolution microscopy at cryo temperatures has also been established using several different approaches.\nWhile fluorescence-derived localization is a key benefit of cryo-CLEM, fluorescence can also be used to bring orthogonal information into cryo-EM images. We recently developed a fusion assay compatible with cryo-CLEM equipment and conditions (Metskas and Briggs, Microscopy & Microanalysis 2019). This development employs auto-quenching by resonant energy transfer to specifically target a function rather than a protein in cryo-CLEM - in this case, adding information on lipid mixing to morphologies from micrographs of influenza virus fusion. However, further methods developments, particularly those involving FRET, are currently hampered by limited characterization of modern fluorophores at cryo-CLEM temperatures (77-100 K).\nHere, we present a study of commonly used synthetic fluorophores and fluorescent proteins, characterizing excitation and emission spectra, singlet state lifetime, and quantum yield at 77 K. We note that 10 nm shifts of the modes are common for both excitation and emission spectra, but are fluorophore specific in magnitude and even in direction. Vibronic coupling and spectral narrowing are visible in all cases characterized, and singlet state lifetimes increase or decrease in a fluorophore-specific manner. Taken together, these data suggest guidelines for choosing cryo-CLEM fluorophores and filter sets, and demonstrate promise for techniques such as FRET in carefully-adapted applications.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/99981, title ="Enzymatic Labeling of Bacterial Proteins for Super-resolution Imaging in Live Cells", author = "Ho, Samuel H. and Tirrell, David A.", journal = "ACS Central Science", volume = "5", number = "12", pages = "1911-1919", month = "December", year = "2019", doi = "10.1021/acscentsci.9b00617", issn = "2374-7943", url = "https://resolver.caltech.edu/CaltechAUTHORS:20191121-100808414", note = "© 2019 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: June 24, 2019; Published: November 21, 2019. \n\nThe authors thank members of the Tirrell laboratory for insightful discussions. S.H.H. thanks Y. Hui, A. Collazo, and R. Stanciauskas for helpful advice. This work was supported by the Joseph J. Jacobs Institute for Molecular Engineering for Medicine and the Center for Environmental Microbial Interactions at the California Institute of Technology. \n\nThe authors declare no competing financial interest.", revision_no = "25", abstract = "Methods that enable the super-resolution imaging of intracellular proteins in live bacterial cells provide powerful tools for the study of prokaryotic cell biology. Photoswitchable organic dyes exhibit many of the photophysical properties needed for super-resolution imaging, including high brightness, photostability, and photon output, but most such dyes require organisms to be fixed and permeabilized if intracellular targets are to be labeled. We recently reported a general strategy for the chemoenzymatic labeling of bacterial proteins with azide-bearing fatty acids in live cells using the eukaryotic enzyme N-myristoyltransferase. Here we demonstrate the labeling of proteins in live Escherichia coli using cell-permeant bicyclononyne-functionalized photoswitchable rhodamine spirolactams. Single-molecule fluorescence measurements on model rhodamine spirolactam salts show that these dyes emit hundreds of photons per switching event. Super-resolution imaging was performed on bacterial chemotaxis proteins Tar and CheA and cell division proteins FtsZ and FtsA. High-resolution imaging of Tar revealed a helical pattern; imaging of FtsZ yielded banded patterns dispersed throughout the cell. The precision of radial and axial localization in reconstructed images approaches 15 and 30 nm, respectively. The simplicity of the method, which does not require redox imaging buffers, should make this approach broadly useful for imaging intracellular bacterial proteins in live cells with nanometer resolution.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/90464, title ="The dormancy-specific regulator, SutA, is intrinsically disordered and modulates transcription initiation in Pseudomonas aeruginosa", author = "Bergkessel, Megan and Babin, Brett M.", journal = "Molecular Microbiology", volume = "112", number = "3", pages = "992-1009", month = "September", year = "2019", doi = "10.1111/mmi.14337", issn = "0950-382X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181029-102517113", note = "© 2019 John Wiley & Sons Ltd. \n\nIssue Online: 10 September 2019; Version of Record online: 10 July 2019; Accepted manuscript online: 28 June 2019; Manuscript accepted: 25 June 2019. \n\nWe thank Ben Ramirez (University of Illinois at Chicago) for helping us with preliminary NMR studies of SutA, Jacqueline Barton (Caltech) for giving us access to her lab to perform experiments involving radioactivity, Nate Glasser for help with HPLC measurements to quantify SutA, Hsiau‐Wei (Jack) Lee and Aimee Marceau (University of California, Santa Cruz) for help with the NMR binding experiment using the Bruker AVIII HD 800 MHz NMR, Weidong Hu (City of Hope) for help with NMR experiments using the Bruker AV III 700 MHz spectrometer, and Julia Kardon and Niels Bradshaw (Brandeis University) and members of the Newman lab for feedback on the project at different stages. MB was supported by a postdoctoral fellowship from the Cystic Fibrosis Foundation (BERGKE16F0). Grants from the NIH (GM067153) to IA and grants from the NIH (R01HL117328 and 1R01AI127850‐01A1) to DKN supported this work. The Proteome Exploration Laboratory is supported by the Beckman Institute and NIH 1S10OD02001301. This work was also supported by the Institute for Collaborative Biotechnologies through grant W911NF‐09‐0001 from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the Government and no official endorsement should be inferred. \n\nThe authors have no conflicts of interest to disclose.", revision_no = "37", abstract = "Though most bacteria in nature are nutritionally limited and grow slowly, our understanding of core processes like transcription comes largely from studies in model organisms doubling rapidly. We previously identified a small protein of unknown function, SutA, in a screen of proteins synthesized in Pseudomonas aeruginosa during dormancy. SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA comprises a central α‐helix with unstructured N‐ and C‐terminal tails, and binds to the β1 domain of RNAP. It activates transcription from the rrn promoter by both the housekeeping sigma factor holoenzyme (Eσ^(70)) and the stress sigma factor holoenzyme (Eσ^S) in vitro, but has a greater impact on Eσ^S. In both cases, SutA appears to affect intermediates in the open complex formation and its N‐terminal tail is required for activation. The small magnitudes of in vitro effects are consistent with a role in maintaining activity required for homeostasis during dormancy. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/97761, title ="Visualizing mechanical stress in artificial protein hydrogels", author = "Baccile, Joshua A. and Tirrell, David Anthony", pages = "PMSE-0414", month = "August", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190812-095412177", note = "© 2019 American Chemical Society.", revision_no = "10", abstract = "Soft materials with made-to-order chem. and phys. properties have significant potential value in fields ranging from regenerative medicine and in vitro cell culture to soft robotics and drug delivery. Artificial protein hydrogels (APHs) are uniquely suited to be designer materials via exploitation of the near limitless possible combinations of functional protein domains that can be used to create novel polymer building blocks with tailored properties. Recent work in model systems has demonstrated APHs can be engineered to improve wound healing, for controlled drug delivery and for the creation of materials with programmed characteristic relaxation times. However, engineering APHs to have enhanced mech. strength and toughness has so far not been possible and remains a crit. obstacle for their clin. deployment and use in other applications. One potential approach to make tougher APHs is by creating them from protein structures encoded with a higher propensity to dissipate energy through unfolding in response to mech. stress. This approach seems promising; however, it is not currently understood how features at the mol. level permeate to the bulk phys. properties of APHs. I will discuss our efforts to address this gap through the development of a fluorescence microscopy method for real-time visualization of protein conformational changes that result from mech. stress induced on an APH. The talk will focus on two key points: (1) Design and synthesis of a tunable force-responsive single-mol. FRET sensor that can be readily incorporated into APHs and other types of hydrogels; and (2) fundamental insights on the mechanism of energy dissipation in protein hydrogels gained from visualizing spatially resolved mech. stress. Lastly, I will briefly discuss an application of visualizing mech. stress in APHs - quantifying cell traction forces in three-dimensional cell culture systems.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/96228, title ="N-Myristoyl Transferase (NMT)-Catalyzed Labeling of Bacterial Proteins for Imaging in Fixed and Live Cells", author = "Ho, Samuel H. and Tirrell, David A.", number = "2012", pages = "315-326", month = "June", year = "2019", doi = "10.1007/978-1-4939-9546-2_16", isbn = "978-1-4939-9545-5", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190610-084517342", note = "© 2019 Springer Science+Business Media, LLC, part of Springer Nature. \n\nFirst Online 04 June 2019.", revision_no = "12", abstract = "Methods for selective protein imaging are critical for elucidating how cells orchestrate fundamental biological processes. We recently developed a chemoenzymatic method to modify bacterial proteins in situ for fluorescence imaging using N-myristoyl transferase (NMT). Target proteins outfitted with an N-terminal NMT recognition sequence are covalently modified with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows for conjugation to cell-permeant fluorophores and imaging by fluorescence microscopy. Here we describe sample preparation and labeling protocols for imaging bacterial proteins in fixed and live cells.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/94094, title ="Award Address (Ronald Breslow Award for Achievement in Biomimetic Chemistry sponsored by the Ronald Breslow Award Endowment). Reinterpreting the Genetic Code: Non-Canonical Amino Acids in Protein Science and Engineering", author = "Tirrell, David Anthony", pages = "BIOL-0198", month = "April", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190325-085114527", note = "© 2019 American Chemical Society.", revision_no = "9", abstract = "The genetic code, elucidated in the 1960s through the work of Nirenberg, Ochoa, Khorana and their coworkers, provides a set of mol. instructions for translating nucleic acids into proteins. Through the efforts of our lab. and others, the code has been \"reinterpreted\" in various ways to allow the participation of an expanded set of amino acids in cellular protein synthesis. These developments have enabled powerful new approaches to protein design and to time-resolved, state-selective, and cell-selective anal. of protein synthesis in complex biol. systems.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/94092, title ="Enzymatic labeling of bacterial proteins for live-cell super-resolution imaging", author = "Ho, Samuel and Tirrell, David Anthony", pages = "BIOL-0187", month = "April", year = "2019", url = "https://resolver.caltech.edu/CaltechAUTHORS:20190325-084657712", note = "© 2019 American Chemical Society.", revision_no = "10", abstract = "Mol. probes that illuminate the subcellular organization of prokaryotic organisms are essential for our understanding of the biol. world. We describe here a strategy for labeling bacterial proteins in situ with azido fatty acids using the enzyme N-myristoyltransferase. Cell-permeant photoswitchable fluorescent dyes are conjugated to target proteins through means of strain-promoted azide-alkyne cycloaddn. to enable super-resoln. imaging in live Escherichia coli cells. We demonstrate the method by labeling and imaging bacterial chemotaxis proteins and cell division proteins. The method provides a general framework for live-cell protein imaging with nanometer resoln.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/90152, title ="Replacement of ProB28 by pipecolic acid protects insulin against fibrillation and slows hexamer dissociation", author = "Fang, Katharine Y. and Lieblich, Seth A.", journal = "Journal of Polymer Science Part A: Polymer Chemistry", volume = "57", number = "3", pages = "264-267", month = "February", year = "2019", doi = "10.1002/pola.29225", issn = "0887-624X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181008-104242240", note = "© 2018 Wiley Periodicals, Inc. \n\nReceived 5 July 2018; Accepted 10 August 2018. \n\nK. Y. Fang and S. A. Lieblich contributed equally to this work. \n\nFunding Information: Amgen; Natural Sciences and Engineering Research Council of Canada (NSERC, PGS‐D); Natural Sciences and Engineering Research Council of Canada; Novo Nordisk Foundation.", revision_no = "13", abstract = "Non‐canonical amino acid mutagenesis was used to examine the biophysical consequences of changing ring size and structure at the single proline site in insulin. Addition of a methylene spacer to the prolyl ring (by replacement of proline by pipecolic acid at position B28) led to an increase in stability and a decrease in the rate of hexamer dissociation. The results of this work illustrate the power of non‐canonical amino acid mutagenesis in the engineering of macromolecular aggregation and protein therapeutics.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/90073, title ="Mechanisms of diffusion in associative polymer networks: evidence for chain hopping", author = "Rapp, Peter B. and Omar, Ahmad K.", journal = "Journal of the American Chemical Society", volume = "140", number = "43", pages = "14185-14194", month = "October", year = "2018", doi = "10.1021/jacs.8b07908", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181001-131358075", note = "© 2018 American Chemical Society. \n\nReceived: July 25, 2018; Published: October 1, 2018.\n\nWe thank Andres Collazo of the Caltech Beckman Imaging Facility for expert assistance with acquisition of FRAP data. This work was supported by grant number DMR-1506483 from the Biomaterials Program of the U.S. National Science Foundation. A.K.O. acknowledges support from the National Science Foundation Graduate Research Fellowship under grant no. DGE-1144469 and an HHMI Gilliam Fellowship. B.R.S. acknowledges support from NIH predoctoral training grant 1T32GM112592 and from the Rosen Center for Bioengineering.\n\nP.B.R. and A.K.O. contributed equally to this work.\n\nThe authors declare no competing financial interest.", revision_no = "21", abstract = "Networks assembled by reversible association of telechelic polymers constitute a common class of soft materials. Various mechanisms of chain migration in associative networks have been proposed; yet there remains little quantitative experimental data to discriminate among them. Proposed mechanisms for chain migration include multichain aggregate diffusion as well as single-chain mechanisms such as “walking” and “hopping”, wherein diffusion is achieved by either partial (“walking”) or complete (“hopping”) disengagement of the associated chain segments. Here, we provide evidence that hopping can dominate the effective diffusion of chains in associative networks due to a strong entropic penalty for bridge formation imposed by local network structure; chains become conformationally restricted upon association with two or more spatially separated binding sites. This restriction decreases the effective binding strength of chains with multiple associative domains, thereby increasing the probability that a chain will hop. For telechelic chains this manifests as binding asymmetry, wherein the first association is effectively stronger than the second. We derive a simple thermodynamic model that predicts the fraction of chains that are free to hop as a function of tunable molecular and network properties. A large set of self-diffusivity measurements on a series of model associative polymers finds good agreement with this model.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/90768, title ="Engineered living materials", author = "Liu, Hanwei and Sim, Seunghyun", pages = "POLY-567", month = "August", year = "2018", url = "https://resolver.caltech.edu/CaltechAUTHORS:20181108-144502922", note = "© 2018 American Chemical Society.", revision_no = "10", abstract = "We are exploring the prepn. and properties of soft materials constituted from mixts. of macromols. and bacterial cells. Issues of interest include the roles of cell identity, cellularity, intercellular interaction, macromol. compn. and concn., and macromol.-cellular adhesion in detg. the phys. and biol. behavior of such systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86231, title ="Glucocorticoid signaling enhances expression of glucose-sensing molecules in immature pancreatic beta-like cells derived from murine embryonic stem cells in vitro", author = "Ghazalli, Nadiah and Wu, Xiaoxing", journal = "Stem Cells and Development", volume = "27", number = "13", pages = "898-909", month = "July", year = "2018", doi = "10.1089/scd.2017.0160", issn = "1547-3287", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180507-075120790", note = "© 2018 Mary Ann Liebert, Inc. publishers. \n\nReceived for publication August 1, 2017; Accepted after revision April 30, 2018; Prepublished on Liebert Instant Online May 2, 2018. \n\nThe authors thank Donna Isbell and Kelley Carpenter from the Animal Research Center of City of Hope for assistance in breeding the GR β-cell KO mice. The authors also thank Dr. Manami Hara for providing MIP-EGFP ES cells. This work was supported, in part, by the National Institutes of Health (R01DK081587 and R01DK099734 to H.T.K. and P30CA33572 to City of Hope). Support from the Oxnard Foundation, Ella Fitzgerald Foundation and the Wanek Family Project of Type 1 Diabetes to H.T.K. is also gratefully acknowledged. N.G. was supported by a predoctoral fellowship as part of an institutional grant to City of Hope from the California Institute for Regenerative Medicine (CIRM). Work at California Institute of Technology was supported by the Department of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program to M.T.K. and by National Science Foundation grant DMR 1506483 to D.A.T. The sponsor did not participate in the study design, collection, analysis, and interpretation of data. \n\nNo competing financial interests exist.", revision_no = "18", abstract = "Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. Here, we aim to identify small molecules that affect immature beta cells. A cell-based assay, employing pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an Insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative RT-PCR analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM D-glucose and stimulated by 17 mM D-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/86959, title ="Incorporation of Non-Canonical Amino Acids into Proteins by Global Reassignment of Sense Codons", author = "Fang, Katharine Y. and Lieblich, Seth A.", number = "1798", pages = "173-186", month = "June", year = "2018", doi = "10.1007/978-1-4939-7893-9_13", isbn = "978-1-4939-7892-2", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180611-083918324", note = "© 2018 Springer Science+Business Media, LLC, part of Springer Nature. \n\nFirst Online: 05 June 2018. \n\nWe thank past and present members of the Tirrell laboratory for their contributions to the development and use of ncAAs in protein science and engineering.", revision_no = "9", abstract = "Non-canonical amino acids are finding increasing use in basic and applied research. Proteins that evolved naturally for biological function did so by exploiting the chemistries of the canonical amino acids; however, when proteins are repurposed for biomedical and pharmacological applications, they are often subject to conditions different from those characteristic of their original biological environments. Non-canonical amino acids can impart properties that are inaccessible within canonical protein sequence space, and can thereby lead to improved or new functionality. We describe simple methods for global replacement of canonical amino acids by their non-canonical counterparts in recombinant proteins made in high yield in bacterial expression hosts. These methods can be used to engineer both chemical and physical properties of recombinant proteins.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/85801, title ="Chemical tools for protein imaging in live bacterial cells", author = "Ho, Samuel and Tirrell, David", pages = "BIOL-297", month = "March", year = "2018", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180412-161333505", note = "© 2018 American Chemical Society.", revision_no = "9", abstract = "Reliable methods to det. the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biol. We\nreport a simple and general technique for imaging bacterial proteins in situ by fluorescence microscopy. The method uses\nthe eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid.\nSubsequent strain-promoted azide-alkyne cycloaddn. allows conjugation of dyes and imaging of tagged proteins by confocal\nfluorescence microscopy. This method is demonstrated through labeling of the chemotaxis proteins Tar and CheA and the cell\ndivision proteins FtsZ and FtsA in Escherichia coli. Distinct spatial patterns for each of these proteins are obsd. in both fixed\nand live cells. Furthermore, we describe a photoswitchable fluorescent reporter to effect super-resoln. imaging of bacterial\nchemotaxis proteins. The methods described herein should prove broadly useful for protein imaging in bacteria.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/85795, title ="Engineering dynamic behavior in protein networks", author = "Dooling, Lawrence and Tirrell, David", pages = "PMSE-65", month = "March", year = "2018", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180412-154255921", note = "© 2018 American Chemical Society.", revision_no = "9", abstract = "In protein-based materials, the amino acid sequence can encode chem. and biol. activity and macroscopic material properties.\nHere, we describe two approaches to engineer the dynamic behavior of protein networks that contain coiled-coil phys.\ncross-links. The first is a protein engineering approach in which we have introduced single point mutations within the coiled-coil\ndomain. Hydrogels prepd. from these protein variants exhibit stress relaxation times that vary from less than 1 s to\ngreater than 1000 s. The second approach to modulate network dynamics uses small hydrophobic ligands that bind within the\npore of the coiled coil. Addn. of ligands including fatty acids and vitamin D stabilizes phys. cross- links and slows network\nrelaxation by up to three orders of magnitude. Together, these approaches demonstrate that the dynamic behavior of\nprotein networks is highly sensitive to the mol. details of the phys. cross-links. As a result, network dynamics can be\nencoded within an amino acid sequence or engineered post- fabrication by formulating protein hydrogels with the appropriate ligands.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/85852, title ="Strategies for controlled bacterial assembly resulting in activation of a quorum-sensing circuit", author = "Kozlowski, Mark and Silverman, Bradley", pages = "BIOT-406", month = "March", year = "2018", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180413-152612878", note = "© 2018 American Chemical Society.", revision_no = "9", abstract = "Bacterial communities show promise in a wide range of applications, such as environmental remediation and multi-step\nbiochem. synthesis. Here we present methods by which bacteria can be assembled in two distinct architectures through\ncontrolled surface display of assocg. proteins (SpyTag and SpyCatcher or proteins contg. coiled-coil peptides including SynZip\n17 and SynZip 18). We can tune the size of the aggregates by addn. of a \"stoichiometric excess\" of one type of cell in an\ninteracting pair, as well as by modulating the amt. of protein displayed on the surface. We further demonstrate reversibility of\nthe SynZip-mediated interactions, permanence of the SpyTag-SpyCatcher mediated clustering, and construction of an\northogonal assembly system using two different promoter families. We have used these systems to make simple aggregates\nas well as more complex core-shell structures, and we trigger quorum sensing behavior by cells within these aggregates at\nlow global cell densities. In sum, the techniques we have developed provide a new level of control of genetically-mediated\nbacterial clustering, and suggest potential applications in the construction of complex bioreactor systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/85404, title ="Programmable viscoelastic matrices from artificial proteins", author = "Dooling, L. J. and Tirrell, D. A.", journal = "Molecular Biology of the Cell", volume = "28", number = "26", pages = "Art. No. P3442", month = "December", year = "2017", issn = "1059-1524", url = "https://resolver.caltech.edu/CaltechAUTHORS:20180321-144348585", note = "© 2017 American Society for Cell Biology. Free via Creative Commons 2 months after publication.", revision_no = "10", abstract = "Extracellular matrix compliance influences cellular adhesion and migration, proliferation and apoptosis,\nand differentiation. Much of our current knowledge of the effects of substrate stiffness on cellular behavior is based on elastic substrates, in particular cross‐linked polyacrylamide hydrogels. Biological tissues, however, are viscoelastic and exhibit stress relaxation and energy dissipation on physiologically relevant timescales. While emerging evidence suggests that these physical properties also influence cellular behavior, materials in which viscoelasticity can be precisely engineered are currently lacking. Here, we describe programmable hydrogel matrices assembled from artificial recombinant proteins designed to be cross‐linked by covalent bonds involving cysteine residues, by association of helical domains as coiled coils, or by both mechanisms. Using these proteins, we construct chemical, physical, and chemical‐physical hydrogel networks that deform elastically or viscoelastically depending on the type of cross‐linking (Dooling et al., Adv. Mater., 2016, 28, 4651–4657). In viscoelastic networks, the amount of stress relaxation is tuned by controlling the ratio of physical cross‐linking to chemical crosslinking, and the timescale for stress relaxation is tuned over five orders of magnitude by single point mutations to the coiled‐coil physical cross‐linking domain (Dooling and Tirrell, ACS Cent. Sci., 2016, 2,\n812–819). The genetic engineering approach also allows biological activity to be encoded directly within\nthe protein sequence in the form of cell‐adhesive domains and proteolytic cleavage sites. The capacity to program the viscoelasticity and biological activity of hydrogel matrices is anticipated to have applications in studying and engineering cell‐matrix interactions.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/83141, title ="Cell-type-specific metabolic labeling of nascent proteomes in vivo", author = "Alvarez-Castelao, Beatriz and Schanzenbächer, Christoph T.", journal = "Nature Biotechnology", volume = "35", number = "12", pages = "1196-1201", month = "December", year = "2017", doi = "10.1038/nbt.4016", issn = "1087-0156", url = "https://resolver.caltech.edu/CaltechAUTHORS:20171113-085844680", note = "© 2017 Macmillan Publishers Limited, part of Springer Nature. \n\nReceived: 12 December 2016; Accepted: 19 October 2017; Published online: 06 November 2017. \n\nWe thank H. Geptin, D. Vogel, N. Fuerst, I. Wüllenweber and F. Rupprecht for excellent technical assistance. We thank E. Noll for the synthesis of ANL and P. Landgraf for the synthesis of the SH-alkyne. We thank S. Garg for her help with FUNCAT and M. Heumueller for his help with some of the experiments. We thank R. Pieaud and S. Junek for their assistance with imaging. We thank F. Kretschmer for his help with the open field analysis. We thank E. Northrup, S. Zeissler, S. Gil Mast and the animal facility of MPI for Brain Research for their excellent support. We thank J. Sanes and J. Chakkalakal for the early generation of a Thy-1 MetRS* mouse. Work in the laboratory of E.M.S. is supported by the Max Planck Society, the European Research Council, DFG CRC 902 and 1080, and the DFG Cluster of Excellence for Macromolecular Complexes. B.A. was supported by a Marie Curie Intra-European Fellowship for career development. C.H. was supported by a Marie Curie Career Integration Grant. D.C.D. is supported by DFG CRC 779 and 854. Research on proteomic labelling at Caltech is supported by the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation. \n\nAuthor Contributions: B.A.-C., C.T.S. and C.H.: conception and design of experiments, acquisition, analysis and interpretation of data. C.G., S.t.D. and A.R.D.: conception and design of experiments, acquisition of data. I.B., B.N.-A. and E.C.: acquisition of data. A.M.: provided reagents. D.D.: provided reagents and advice on experiments. D.A.T.: conception and design of experiments. J.D.L.: acquisition of data, analysis and interpretation of data. E.M.S.: conception and design of experiments, analysis and interpretation of data, drafting, writing and revising the article. All authors contributed to the writing and revision of the manuscript. \n\nThe authors declare no competing financial interests.", revision_no = "48", abstract = "Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/82764, title ="Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms", author = "Babin, Brett M. and Atangcho, Lydia", journal = "mBio", volume = "8", number = "5", pages = "Art. No. e01593-17", month = "October", year = "2017", doi = "10.1128/mBio.01593-17", issn = "2150-7511", url = "https://resolver.caltech.edu/CaltechAUTHORS:20171030-071731344", note = "Copyright © 2017 Babin et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International license. \n\nReceived 6 September 2017 . Accepted 20 September 2017. Published 24 October 2017. \n\nWe thank Geoff Smith and Roxana Eggleston-Rangel for assistance with liquid chromatography-tandem mass spectrometry and Mustafa Fazli for assistance with flow cell biofilm cultures. We appreciate the constructive feedback on the manuscript provided by members of the D.K.N. and D.A.T. laboratories. D.K.N. helped support this research with grants from the Howard Hughes Medical Institute and NIH (5R01HL117328-03). T.T.-N. was supported by a grant from the Danish Council for Independent Research (DFF–1323-00177). M.B.V.E. was supported by the Netherlands Organization for Scientific Research (Rubicon fellowship 680-50-1407). The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation through grant GBMF775, the Beckman Institute, and NIH S10RR029594. This work was also supported by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office. The content of the information does not necessarily reflect the position or the policy of the U.S. Government, and no official endorsement should be inferred.", revision_no = "11", abstract = "Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/81405, title ="Protein-Mediated Colloidal Assembly", author = "Obana, Maiko and Silverman, Bradley R.", journal = "Journal of the American Chemical Society", volume = "139", number = "40", pages = "14251-14256", month = "October", year = "2017", doi = "10.1021/jacs.7b07798", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170913-101124804", note = "© 2017 American Chemical Society. \n\nReceived: August 1, 2017; Published: September 12, 2017. \n\nWe thank Trudy Padmore for technical assistance with particle conjugation and Andres Collazo for assistance with confocal microscopy. This work was supported by Defense Advanced Research Projects Agency Biological Robustness in Complex Settings Contract HR001-15-C-0093. M.O is supported by the Nakajima Foundation. B.S. is supported by NIH Training Grant 1T32GM112592 and by the Rosen Center for Bioengineering. Imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. \n\nAuthor Contributions: M.O and B.R.S. contributed equally to this work. \n\nThe authors declare the absence of any competing financial interests.", revision_no = "32", abstract = "Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein–protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein–protein interactions can be used to assemble complex core–shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/82307, title ="SutA is a Global Transcriptional Regulator Operating during Slow Growth in Pseudomonas Aeruginosa", author = "Bergkessel, M. and Newman, D. K.", journal = "Pediatric Pulmonology", volume = "52", number = "S47", pages = "S366-S367", month = "September", year = "2017", doi = "10.1002/ppul.23840", issn = "8755-6863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20171012-102706675", note = "© 2017 Wiley Periodicals, Inc. \n\nIssue online: 19 September 2017; Version of record online: 19 September 2017.", revision_no = "12", abstract = "Chronic Pseudomonas aeruginosa infections, a major cause of morbidity and mortality for CF patients, are highly resistant to antibiotic treatment, and many lines of evidence suggest that this is due in part to slow-growing\nstates imposed by the lung environment. In previously published work (Babin BM, et al. Proc Natl Acad Sci 2016;113(5):E597-605), we identified a novel RNA polymerase-binding protein that is post-transcriptionally upregulated under such slow-growth conditions. We named this small,\nacidic protein SutA, and showed using RNA-Seq and ChIP-Seq that it associates with RNA polymerase (RNAP) at many genomic regions, and tends to positively affect transcript levels for genes with which it associates. The genes that are affected include ribosomal RNA and protein genes, and other genes with roles in metabolism and stress survival. Furthermore, we showed that deletion of the sutA gene confers a disadvantage on cells subjected to fluctuations between growth-arrested and growth-promoting states. Thus,\nSutA may contribute to survival of Pseudomonas aeruginosa in environments like the CF lung, where oxygen and nutrient availability are likely to be heterogeneous in time and space.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/78131, title ="4S-Hydroxylation of insulin at ProB28 accelerates hexamer dissociation and delays fibrillation", author = "Lieblich, Seth A. and Fang, Katharine Y.", journal = "Journal of the American Chemical Society", volume = "139", number = "25", pages = "8384-8387", month = "June", year = "2017", doi = "10.1021/jacs.7b00794", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170612-160413860", note = "© 2017 American Chemical Society. \n\nReceived: January 24, 2017. Published: June 9, 2017. \n\nWe thank J. T. Kaiser, P. Nikolovski, S. Russi, S. Virgil, M. Shahgholi, A. Lakshmanan, and the scientific staff of Beamline 12-2 at the Stanford Synchrotron Radiation Laboratory for assistance. We thank W. Glenn, A. Mahdavi, and T. Hoeg-Jensen for discussions. The work was supported by the Novo Nordisk Foundation. Fellowships from Amgen and from the Natural Sciences and Engineering Research Council of Canada (NSERC, PGS-D) provided partial support for S.A.L. and K.Y.F., respectively. J.K.B.C. acknowledges support of the Resnick Sustainability Institute (Caltech). \n\nThe authors declare the following competing financial interest(s): S.A.L., K.Y.F., and D.A.T are inventors on a related patent application.", revision_no = "29", abstract = "Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of non-canonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/77188, title ="A Fluorescence in Situ Hybridization Method To Quantify mRNA Translation by Visualizing Ribosome–mRNA Interactions in Single Cells", author = "Burke, Kelly S. and Antilla, Katie A.", journal = "ACS Central Science", volume = "3", number = "5", pages = "425-433", month = "May", year = "2017", doi = "10.1021/acscentsci.7b00048", issn = "2374-7943", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170504-090316077", note = "© 2017 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: January 25, 2017; Published: May 3, 2017. \n\nWe thank the National Science Foundation Graduate Research Fellowship Program (NSF GRFP, Grant Number 1144469), the Rose Hills Foundation (Caltech SURF), the German Research Foundation (Grant No. MI 1315/4), and the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation for support of this work. We thank H. M. T. Choi and N. A. Pierce for suggesting the use of a linker probe carrying an HCR initiator as a mechanism for selectively generating signal from cognate probe pairs colocalized by targets in the sample. We thank Florian Mueller for development of FISH-quant and the Broad Institute for development of Cell Profiler for image analysis. We thank Johannes Stegmaier and the Center for Advanced Methods in Biological Image Analysis at the Beckman Institute (CAMBIA) for adapting the XPIWIT software tool for analysis of nuclear transcripts. We thank Andres Collazo for assistance with the LSM 800 confocal microscope in the Biological Imaging Facility of the Beckman Institute at Caltech.", revision_no = "25", abstract = "Single-molecule fluorescence in situ hybridization (smFISH) is a simple and widely used method to measure mRNA transcript abundance and localization in single cells. A comparable single-molecule in situ method to measure mRNA translation would enable a more complete understanding of gene regulation. Here we describe a fluorescence assay to detect ribosome interactions with mRNA (FLARIM). The method adapts smFISH to visualize and characterize translation of single molecules of mRNA in fixed cells. To visualize ribosome–mRNA interactions, we use pairs of oligonucleotide probes that bind separately to ribosomes (via rRNA) and to the mRNA of interest, and that produce strong fluorescence signals via the hybridization chain reaction (HCR) when the probes are in close proximity. FLARIM does not require genetic manipulation, is applicable to practically any endogenous mRNA transcript, and provides both spatial and temporal information. We demonstrate that FLARIM is sensitive to changes in ribosome association with mRNA upon inhibition of global translation with puromycin. We also show that FLARIM detects changes in ribosome association with an mRNA whose translation is upregulated in response to increased concentrations of iron.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/77659, title ="Cell-selective proteomic analysis of host-microbe interactions using Bio-orthogonal Noncanonical Amino Acid Tagging (BONCAT)", author = "Stone, Shannon and Shon, Judy", pages = "BIOT-467", month = "April", year = "2017", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170523-110036625", note = "© 2017 American Chemical Society.", revision_no = "12", abstract = "While proteomic studies of microbes in culture have provided many important biol. insights, there is a need for more global analyses of their behavior during interactions within mammalian hosts. However, abundant host tissues often dominate and obstruct system-wide profiling of proteins expressed by microbes in mouse models. We have adapted bio-orthogonal non-canonical amino add tagging (BONCAT), a chemoproteomic tool for detecting newly-synthesized proteins in complex biol. systems, to cell-selectively label and identify microbial proteins while infecting or colonizing a live mouse. Diverse species of\nmicrobes have been labeled using this technique, Including methicillin-resistant Staphylococcus aureus (MRSA), and the human gut commensal, Bacteroides fragilis. When coupled to click. chem., this cell-selective technique can be used to visualize proteomes in space and time using fluorescence microscopy, or to enrich and identify proteins using mass spectrometry. In addn. to confirming well-established virulence factors in MRSA, we found a previously unidentified factor, and the top BONCAT hit, to play a crit. role in skin infection models. Furthermore, we are exploring combining this method with passive CLARITY\ntechniques to visualize microbial population dynamics within mice. Our work demonstrates the power of unbiased\nchemoproteomic labeling in vivo. A08.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/74522, title ="Analysis and control of chain mobility in protein hydrogels", author = "Rapp, Peter B. and Omar, Ahmad K.", journal = "Journal of the American Chemical Society", volume = "139", number = "10", pages = "3796-3804", month = "March", year = "2017", doi = "10.1021/jacs.6b13146", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170224-091637468", note = "© 2017 American Chemical Society. \n\nReceived: December 30, 2016; Published: February 22, 2017. \n\nWe thank Steven Olsen and the Division of Chemistry and Chemical Engineering Instrument Shop for machining sample holders for the FRAP experiments. We also thank David Koos, Andres Collazo and the Biological Imaging Facility of the Caltech Beckman Institute for training and assistance in operating the confocal microscope. We thank John Bagert, Lawrence Dooling, and Cole DeForest for helpful discussions during the preparation of the manuscript. \n\nThis work was supported by grant number DMR-1506483 from the Biomaterials Program of the U. S. National Science Foundation. A.K.O. acknowledges support by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1144469 and an HHMI Gilliam Fellowship. \n\nThe authors declare no competing financial interest.", revision_no = "19", abstract = "Coiled-coil domains can direct the assembly of protein block copolymers into physically crosslinked, viscoelastic hydrogels. Here we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a 3-state “hopping” model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the 3-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/73620, title ="BONCAT enables time-resolved analysis of protein synthesis in native plant tissue", author = "Glenn, Weslee S. and Stone, Shannon E.", journal = "Plant Physiology", volume = "173", number = "3", pages = "1543-1553", month = "March", year = "2017", doi = "10.1104/pp.16.01762", issn = "0032-0889", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170123-111643957", note = "© 2017 American Society of Plant Biologists. Free via OPEN. \n\nReceived November 17, 2016; Accepted January 14, 2017; First Published on January 19, 2017. \n\nWe would like to thank the laboratory of Professor Elliot Meyerowitz, especially Arnavaz Garda and Dr. Paul Tarr, for seeds, space in growth chambers and helpful discussions. We would also like to thank Roxana Eggleston-Rangel for helpful discussions on sample preparation for mass spectrometry. \n\nThis work was supported by the Gordon and Betty Moore Foundation through grant GBMF2809. W.S.G. was supported by a National Research Council Ford Foundation Post-Doctoral\nFellowship and a United Negro College Fund/Merck Foundation Post-Doctoral Fellowship. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation, through Grant GBMF775, the Beckman Institute and the NIH through Grant 1S10RR029594-01A1. \n\nAuthor Contributions: W.S.G. conceived of and implemented the project, and performed most of the experiments with contributions from all other authors. S.H.H., J.B-S and W.S.G. designed and conducted gel imaging and immunoblotting experiments. A.M., S.H. and W.S.G. designed and ran the mass spectrometry experiments. S.E.S., M.J.S., S.H., J. B-S and W.S.G. conducted all bioinformatic analyses of the mass spectrometry data. S.E.S., M.J.S. and W.S.G. constructed figures with input from all other authors. W.S.G, J.B-S and D.A.T. wrote the article with valuable contributions from all authors.", revision_no = "24", abstract = "Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal non-canonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis thaliana seedlings. BONCAT relies on the translational incorporation of a non-canonical amino acid (ncAA) probe into cellular proteins. In this study, the probe is the methionine surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse, and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry (LC-MS) to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and post-translational regulation in plants.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/74775, title ="Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue", author = "Glenn, Weslee S. and Stone, Shannon E.", journal = "Plant Physiology", volume = "173", number = "3", pages = "1543-1553", month = "March", year = "2017", doi = "10.1104/pp.16.01762", issn = "0032-0889", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170306-102953244", note = "© 2017 American Society of Plant Biologists. Article can be viewed without a subscription. \n\nReceived November 17, 2016; accepted January 14, 2017; published January 19, 2017. \n\nWe thank the laboratory of Professor Elliot Meyerowitz, especially Arnavaz Garda and Dr. Paul Tarr, for seeds, space in growth chambers, and helpful discussions. We also thank Roxana Eggleston-Rangel for helpful discussions on sample preparation for mass spectrometry. \n\nThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David A. Tirrell (tirrell@caltech.edu). \n\nW.S.G. conceived of and implemented the project and performed most of the experiments with contributions from all other authors; S.H.H., J.B.-S., and W.S.G. designed and conducted gel imaging and immunoblotting experiments; A.M., S.H., and W.S.G. designed and ran the mass spectrometry experiments; S.E.S., M.J.S., S.H., J.B.-S., and W.S.G. conducted all bioinformatic analyses of the mass spectrometry data; S.E.S., M.J.S., and W.S.G. constructed figures with input from all other authors; W.S.G., J.B.-S., and D.A.T. wrote the article with valuable contributions from all authors. \n\nThis work was financially supported by the Gordon and Betty Moore Foundation through grant GBMF2809. W.S.G. was supported by a National Research Council Ford Foundation Post-Doctoral Fellowship and a United Negro College Fund/ Merck Foundation Post-Doctoral Fellowship. The Proteome Exploration Laboratory is supported by the Gordon and Betty Moore Foundation, through grant GBMF775, the Beckman Institute, and the NIH through grant 1S10RR029594-01A1. \n\nAccession Numbers: Raw mass spectrometry files have been uploaded to the Japan Proteome standard Repository under accession number PXD005577.", revision_no = "33", abstract = "Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/73527, title ="Cell-selective proteomics for biological discovery", author = "Stone, Shannon E. and Glenn, Weslee S.", journal = "Current Opinion in Chemical Biology", volume = "36", pages = "50-57", month = "February", year = "2017", doi = "10.1016/j.cbpa.2016.12.026", issn = "1367-5931", url = "https://resolver.caltech.edu/CaltechAUTHORS:20170117-150138453", note = "© 2017 Elsevier B.V. \n\nAvailable online 12 January 2017. \n\nCaltech research on cell-specific proteomic analysis has been supported by NIH grants R01-GM062523 and R21-AI121890, and by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office.", revision_no = "17", abstract = "Cells alter the proteome to respond to environmental and developmental cues. Global analysis of proteomic responses is of limited value in heterogeneous environments, where there is no ‘average’ cell. Advances in sequencing, protein labeling, mass spectrometry, and data analysis have fueled recent progress in the investigation of specific subpopulations of cells in complex systems. Here we highlight recently developed chemical tools that enable cell-selective proteomic analysis of complex biological systems, from bacterial pathogens to whole animals.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/72050, title ="Chemoenzymatic Labeling of Proteins for Imaging in Bacterial Cells", author = "Ho, Samuel H. and Tirrell, David A.", journal = "Journal of the American Chemical Society", volume = "138", number = "46", pages = "15098-15101", month = "November", year = "2016", doi = "10.1021/jacs.6b07067", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20161116-100355067", note = "© 2016 American Chemical Society. \n\nReceived: July 16, 2016; Published: November 10, 2016. \n\nThe authors thank B. M. Babin, S. E. Stone, L. J. Dooling, K. P. Yuet, K. S. Burke, J. D. Bagert, and C. Kulkarni for insightful discussions. S.H.H. thanks D. K. Romney, S. Brinkmann-Chen, A. Collazo, S. Hess, A. Moridan, M. Shahgholi, C. Neubauer, and N. Dalleska for advice. S.H.H. was supported in part by a National Science Foundation Graduate Research Fellowship. This work was supported by the Caltech Center for Environmental Microbial Interactions, the Jacobs Institute for Molecular Engineering for Medicine, and the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the U.S. Army Research Office. \n\nThe authors declare no competing financial interest.", revision_no = "24", abstract = "Reliable methods to determine the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biology. We describe here a simple and general technique for imaging of bacterial proteins in situ by fluorescence microscopy. The method uses the eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows conjugation of dyes and imaging of tagged proteins by confocal fluorescence microscopy. We demonstrate the method by labeling the chemotaxis proteins Tar and CheA and the cell division proteins FtsZ and FtsA in Escherichia coli. We observe distinct spatial patterns for each of these proteins in both fixed and live cells. The method should prove broadly useful for protein imaging in bacteria.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/71384, title ="Engineering the Dynamic Properties of Protein Networks through Sequence Variation", author = "Dooling, Lawrence J. and Tirrell, David A.", journal = "ACS Central Science", volume = "2", number = "11", pages = "812-819", month = "November", year = "2016", doi = "10.1021/acscentsci.6b00205", issn = "2374-7943", url = "https://resolver.caltech.edu/CaltechAUTHORS:20161024-112450940", note = "© 2016 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: July 21, 2016. Publication Date (Web): October 18, 2016. \n\nThis work was supported by Grant Number DMR-1506483 from the Biomaterials Program of the U.S. National Science Foundation. We thank Professor Julia Kornfield for extensive access to the ARES-RFS rheometer and Dr. Mona Shahgholi of the Mass Spectrometry Facility of the Division of Chemistry and Chemical Engineering at Caltech for assistance in measuring protein molar masses. \n\nThe authors declare no competing financial interest.", revision_no = "17", abstract = "The dynamic behavior of macromolecular networks dominates the mechanical properties of soft materials and influences biological processes at multiple length scales. In hydrogels prepared from self-assembling artificial proteins, stress relaxation and energy dissipation arise from the transient character of physical network junctions. Here we show that subtle changes in sequence can be used to program the relaxation behavior of end-linked networks of engineered coiled-coil proteins. Single-site substitutions in the coiled-coil domains caused shifts in relaxation time over 5 orders of magnitude as demonstrated by dynamic oscillatory shear rheometry and stress relaxation measurements. Networks with multiple relaxation time scales were also engineered. This work demonstrates how time-dependent mechanical responses of macromolecular materials can be encoded in genetic information.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/71817, title ="Microbuckling of Fibrous Matrices Enables Long Range Cell Mechanosensing", author = "Burkel, Brian and Lesman, Ayelet", volume = "6", pages = "135-141", month = "September", year = "2016", doi = "10.1007/978-3-319-41351-8_19", issn = "2191-5644", isbn = "978-3-319-41350-1", url = "https://resolver.caltech.edu/CaltechAUTHORS:20161108-141109524", note = "© 2017 The Society for Experimental Mechanics, Inc. \n\nFirst Online: 21 September 2016. \n\nThis work was funded by grants from the National Science Foundation (Division of Materials Research 0520565 and 1206121) and from the California Institute for Regenerative Medicine (RB5-07398). J.N. was supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1144469. A.L. was supported in part by a Rothschild Foundation fellowship.", revision_no = "20", abstract = "When biological cells migrate, divide, and invade, they push and pull on individual fibers of the matrix surrounding them. The resulting fiber displacements are neither uniform nor smooth; rather, displacements localize to form dense fibrous bands that span from one cell to another. It is thought that these bands may be a mechanism by which cells can sense their neighbors, but this hypothesis remains untested, because the mechanism for band formation remains unknown. Using digital volume correlation, we measure the displacements induced by contractile cells embedded in a fibrous matrix. We find that cell-induced displacements propagate over a longer range than predicted by linear elasticity. To explain the long-range propagation of displacements, we consider the effect of buckling of individual matrix fibers, which generates a nonlinear stress-strain relationship. We show that fiber buckling is the mechanism that causes the displacements to propagate over a long range and the bands to form between nearby cells. The results thus show that buckling of individual fibers provides a mechanism by which cells may sense their distant neighbors mechanically.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/70322, title ="Genetic programming of molecular, cellular and materials assembly", author = "Tirrell, David", pages = "POLY-461", month = "August", year = "2016", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160913-160552025", note = "© 2016 American Chemical Society.", revision_no = "11", abstract = "The expression of artificial genes allows programming of mol. structure and intermol. interactions. This lecture will\nexplore the use of programmed intermol. interactions to control the assembly of macromol. networks and microbial\nconsortia.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/66242, title ="Programming Molecular Association and Viscoelastic Behavior in Protein Networks", author = "Dooling, Lawrence J. and Buck, Maren E.", journal = "Advanced Materials", volume = "28", number = "23", pages = "4651-4657", month = "June", year = "2016", doi = "10.1002/adma.201506216", issn = "0935-9648", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160418-105044935", note = "© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. \n\nReceived: December 14, 2015. Revised: January 28, 2016; First published: 9 April 2016. \n\nThis work was supported by the Biomaterials Program of the U. S. National Science Foundation through grant DMR1506483, and by the Gordon and Betty Moore Foundation through grant GBMF2809. Peter Rapp provided PEP protein for rheological measurements. The authors thank Julia Kornfield for allowing extensive access to the rheometer in her laboratory and Amy Fu for providing helpful advice on rheological measurements. Mona Shahgholi in the mass spectrometry facility of Caltech’s Division of Chemistry and Chemical Engineering offered expert assistance in the determination of protein molar masses.", revision_no = "18", abstract = "A set of recombinant artificial proteins that can be cross-linked, by either covalent bonds or association of helical domains or both, is described. The designed proteins can be used to construct molecular networks in which the mechanism of cross-linking determines the time-dependent responses to mechanical deformation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/68838, title ="In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas", author = "Tremblay, Jacob R. and LeBon, Jeanne M.", journal = "Journal of Visualized Experiments", number = "112", pages = "Art. No. e54016", month = "June", year = "2016", doi = "10.3791/54016", issn = "1940-087X", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160705-152413540", note = "© 2016 JoVE. \n\nDate published 6/10/2016, Issue 112. \n\nWe thank Lucy Brown and Alexander Spalla from the Analytical Cytometry Core at City of Hope for assistance in sorting. This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., and U01DK089533 to A.D.R., and by National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T. Supports from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech to D.A.T., and those from Oxnard Foundation and Ella Fitzgerald Foundation to H.T.K. are also gratefully acknowledged. \n\nThis work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., and U01DK089533 to A.D.R., and by National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T. Supports from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech to D.A.T., and those from Oxnard Foundation and Ella Fitzgerald Foundation to H.T.K. are also gratefully acknowledged. Research reported in this publication included work performed in the Analytical Cytometry Core and Light Microscopy Digital Imaging Core supported by the National Cancer Institute of the National Institutes of Health under award number P30CA33572. \n\nStudy sponsor: The sponsor did not participate in the study design, collection, analysis, or interpretation of data. \n\nThe authors have nothing to disclose.", revision_no = "21", abstract = "Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/65873, title ="Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis", author = "Mahdavi, Alborz and Hamblin, Graham D.", journal = "Journal of the American Chemical Society", volume = "138", number = "13", pages = "4278-4281", month = "April", year = "2016", doi = "10.1021/jacs.5b08980", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160404-075529150", note = "© 2016 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nReceived: August 24, 2015. Publication Date (Web): March 18, 2016. \n\nWe are grateful for financial support by National Institutes of Health grant NIH R01 GM062523 and the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation, the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from U.S. Army Research Office. M.S. and S.H. were supported by the Gordon and Betty Moore Foundation through grant GMBF775 and NIH grant 1S10RR029594-01A1. A.M. was supported by a scholarship from the National Science and Engineering Research Council of Canada and by a postgraduate scholarship from the Donna and Benjamin M. Rosen Center for Bioengineering at Caltech. G.D.H. was supported by a postdoctoral fellowship from the National Science and Engineering Research Council of Canada. \n\nThe authors declare no competing financial interest.", revision_no = "37", abstract = "Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNA^(Met) in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/65787, title ="Bio-orthogonal chemistry enables proteomic analysis of Staphylococcus aureus during mammalian infection", author = "Stone, Shannon and Tirrell, David", pages = "BIOT-189", month = "March", year = "2016", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160331-085640195", note = "© 2016 American Chemical Society.", revision_no = "11", abstract = "We report on a new tool to study proteins synthesized by Staphylococcus aureus during infection of a live mouse.\nTraditional proteomics approaches are often unsuitable to study pathogens in vivo because highly abundant host proteins\npredominate. To circumvent this issue, we engineered several strains of S. aureus to have the ability to incorporate a noncanonical\namino acid with a bio- orthogonal handle. Only these engineered strains of S. aureus can incorporate this amino\nacid into newly synthesized proteins, thus rendering these proteins chem. distinct from the host proteome, and allowing us to\nselectively tag and enrich for pathogenic proteins. Before enrichment of proteins tagged in vivo, we found relatively few S.\naureus proteins in samples isolated from a skin infection model. After enrichment, we were able to identify 4x more proteins\nin total, with far greater spectra and sequence coverage per protein. We compared enriched samples from S. aureus labeled\nin vivo to the proteome synthesized prior to infection (in vitro) . Over 1000 proteins were found in the in vitro lysates, while\n484 were identified in the in vivo samples. These results represent the highest no. of S. aureus proteins identified in vivo to\ndate and, in addn. to confirming many previously identified virulence factors, generate new candidates to examine for\ncontributions to virulence.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/65854, title ="Coupling molecular recognition and viscoelastic behavior in engineered protein hydrogels", author = "Tirrell, David and Dooling, Lawrence", pages = "PMSE-135", month = "March", year = "2016", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160401-130603427", note = "© 2016 American Chemical Society.", revision_no = "10", abstract = "Protein gels are of interest as subjects of fundamental study and as matrixes for cell transplantation. This\nlecture will describe some recent expts. directed toward programming the viscoelastic behavior of artificial\nprotein hydrogels through sequence-dependent control of interchain interactions.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/65790, title ="Non-canonical amino acids as tools for protein medicinal chemistry", author = "Tirrell, David and Fang, Katherine", pages = "PRES-77", month = "March", year = "2016", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160331-090004894", note = "© 2016 American Chemical Society.", revision_no = "11", abstract = "The engineering of therapeutic proteins has been advanced enormously by methods for site-directed mutagenesis. This lecture\nwill describe an alternative, complementary approach, in which non- canonical amino acids are used to engineer protein-protein\ninteractions, protein solvation and chain trajectory, with the objective of controlling the stability and pharmacokinetics\nof protein therapeutics.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/62633, title ="Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi", author = "Bagert, John D. and van Kessel, Julia C.", journal = "Chemical Science", volume = "7", number = "3", pages = "1797-1806", month = "March", year = "2016", doi = "10.1039/c5sc03340c", issn = "2041-6520", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151207-090454052", note = "© 2015 The Royal Society of Chemistry. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. \n\nReceived 4th September 2015; Accepted 17th November 2015; First published online 23 Nov 2015. \n\nWe thank Annie Moradian and Roxana Eggleston-Rangel for technical support with LC-MS/MS experiments. This work was supported by the Howard Hughes Medical Institute, National Institutes of Health (NIH) grant 5R01GM065859 and National Science Foundation (NSF) grant MCB-0343821 to BLB, and by NIH Grant R01GM062523 and the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office to DAT. SH and MJS were supported by the Gordon and Betty Moore Foundation, through Grant GBMF775, and the Beckman Institute. The content of the paper does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. \n\nThis article is part of themed collection: ISACS16: Challenges in Chemical Biology.", revision_no = "24", abstract = "Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors. We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/65786, title ="What click chemistry has taught us about cellular protein synthesis", author = "Tirrell, David and Babin, Brett", pages = "CARB-111", month = "March", year = "2016", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160331-085414342", note = "© 2016 American Chemical Society.", revision_no = "13", abstract = "Click chem. has provided powerful new probes for the study of biol. processes. This lecture will describe the use of clickable\namino acids as probes of protein synthesis in complex biol. systems, including dormant bacterial cultures, mouse infection\nmodels, and the nematode Caenorhabditis elegans.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63789, title ="SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa", author = "Babin, Brett M. and Bergkessel, Megan", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "113", number = "5", pages = "E597-E605", month = "February", year = "2016", doi = "10.1073/pnas.1514412113 ", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160120-081453238", note = "© 2015 National Academy of Sciences. \n\nEdited by Lucia B. Rothman-Denes, The University of Chicago, Chicago, IL, and approved December 17, 2015 (received for review July 21, 2015). \n\nPublished online before print January 19, 2016. \n\nWe thank Geoff Smith and Roxana Eggleston-Rangel for technical assistance with liquid chromatography–tandem mass spectrometry and Dr. Igor Antoshechkin for assistance with sequencing. We thank Dr. Olaf Schneewind for his gift of the anti-RpoA antibody. We appreciate constructive feedback on the manuscript from members of the D.K.N. and D.A.T. laboratories and Richard Gourse, as well as helpful comments from the editor and reviewers. This work was supported by NIH Grants 5R01HL117328-03 (to D.K.N.) and 1S10RR029594-01A1 (to S.H.), the Institute for Collaborative Biotechnologies through US Army Research Office Grant W911NF-09-0001 (to D.A.T.), Howard Hughes Medical Institute (HHMI), and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at California Institute of Technology (Caltech). The Proteome Exploration Laboratory (M.J.S., A.M., and S.H.) was supported by Gordon and Betty Moore Foundation Grant GBMF775 and by the Beckman Institute at Caltech. D.K.N. is an HHMI Investigator. \n\nAuthor contributions: B.M.B., M.B., M.J.S., A.M., S.H., D.K.N., and D.A.T. designed research; B.M.B. and M.B. performed research; B.M.B., M.B., M.J.S., S.H., D.K.N., and D.A.T. analyzed data; and B.M.B., M.B., M.J.S., S.H., D.K.N., and D.A.T. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nData deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE66181). \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1514412113/-/DCSupplemental.", revision_no = "38", abstract = "Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/66481, title ="Grand Challenges in Chemistry for 2016 and Beyond", author = "Bertozzi, Carolyn R. and Chang, Christopher J.", journal = "ACS Central Science", volume = "2", number = "1", pages = "1-3", month = "January", year = "2016", doi = "10.1021/acscentsci.6b00010", issn = "2374-7943", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160426-105420652", note = "© 2016 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. \n\nPublished: January 27, 2016.", revision_no = "10", abstract = "When several ACS Central Science Editors met\nfor dinner at the 2015 Pacifichem meeting,\nconversation turned to the grand challenges\nfacing science and society, and those we felt chemistry was in a\nunique position to solve. With the New Year, we thought we\nwould share our ideas in our first editorial of 2016. The arenas\nin which we see chemistry having the largest influence are the\nmolecular bases of disease and aging, alternative energy\nadvancement, and the conservation of our elemental resources.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63344, title ="Cells with surface expression of CD133^(high)CD71^(low) are enriched for tripotent colony-forming progenitor cells in the adult murine pancreas", author = "Jin, Liang and Gao, Dan", journal = "Stem Cell Research", volume = "16", number = "1", pages = "40-53", month = "January", year = "2016", doi = "10.1016/j.scr.2015.11.015", issn = "1873-5061", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160104-143534788", note = "© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). \n\nReceived 11 July 2014. Received in revised form 7 November 2015. Accepted 25 November 2015. Available online 30 November 2015. \n\nThis work is supported in part by the National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K. and U01DK089533 to A.D.R., National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T., and Office of Naval Research ONR-N00014-02-1 0958 and NSF-DBI-9970143 to the Electron Microscopy Core facility at City of Hope. Support from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech is also gratefully acknowledged. L.J. is supported by the National High Technology Research and Development Program of China (863 Program, No. 2015AA020314), Excellent Youth Foundation of Jiangsu Scientific Committee (BK20140029), the Fundamental Research Funds for the Central Universities (Z114037), Priority Academic Program Development of Jiangsu Higher Education Institutions, and the National Natural Science Foundation of China (No. 81570696). \n\nResearch reported in this publication included work performed in the Analytical Cytometry Core and Light Microscopy Digital Imaging Core supported by the National Cancer Institute of the National Institutes of Health under award number P30CA33572. \n\nThe sponsor did not participate in the study design, collection, analysis, or interpretation of data. \n\nAuthor Responsibilities: Liang Jin, Dan Gao: study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript. \n\nTao Feng, Angela Luo, Jeffery Rawson, Janine Quijano, Jing Chai, Lena Wedeken, Nadiah Ghazalli, Jasper Hsu, Jeanne LeBon, Stephanie Walker, Jacob Tremblay: acquisition of data, analysis and interpretation of data. \n\nAlborz Mahdavi, Hung-Ping Shih: technical or material support. \n\nDavid A. Tirrell, Arthur D. Riggs: technical or material support, critical revision of the manuscript for important intellectual content, obtained funding. \n\nHsun Teresa Ku: study concept and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, obtained funding. \n\nThe authors have nothing to disclose.", revision_no = "13", abstract = "Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2–4 month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~ 1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133^(high)CD71^(low) cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133^(high)CD71^(low) cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133^(high)CD71^(low) cells gave rise to colonies that contained duct, acinar, and Insulin+ Glucagon+ double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin+ Glucagon− cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133^(high)CD71^(low) cells. These CD133^(high)CD71^(low) cells will enable future studies of putative adult pancreas stem cells in vivo.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/62117, title ="Two-Site Internally Cooperative Mechanism for Enzyme Kinetics in a Hydrogel Forming Recombinant Protein", author = "Zehner, Nicholas A. and Dietrick, Scott M.", journal = "Biomacromolecules", volume = "16", number = "11", pages = "3651-3656", month = "November", year = "2015", doi = "10.1021/acs.biomac.5b01110", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151116-100832486", note = "© 2015 American Chemical Society. \n\nReceived: August 17, 2015; Published: September 24, 2015. \n\nThe authors declare no competing financial interest.", revision_no = "10", abstract = "A recombinant protein, ATCTA, consisting of three domains, α-helix (A), thrombin cleavage site (T), and water-soluble coil (C), forms hydrogels via the self-association of its flanking α-helices into tetrameric bundles, which act as cross-links for the hydrogel network. In the presence of thrombin, the hydrogel degrades due to the thrombin cleavage sites. To better understand the proteolysis reaction in ATCTA, we performed a series of kinetic experiments on the proteins ATC, CTA, CTATC, and ATCTA. The K_M and k_(cat) of ATC and CTA were determined to be 88 ± 5 μM and 6.4 ± 0.1 s^(–1) and 91 ± 9 μM and 6.1 ± 0.1 s^(–1), respectively. Using these kinetic parameters, a model based on a two-site internally cooperative mechanism was developed to describe the kinetics of proteins containing two cleavage sites. This model was then validated by comparing predicted results with kinetic data from the proteolysis of ATCTA.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/58067, title ="Quantifying cell-induced matrix deformation in three dimensions based on imaging matrix fibers", author = "Notbohm, Jacob and Lesman, Ayelet", journal = "Integrative Biology", volume = "7", number = "10", pages = "1186-1195", month = "October", year = "2015", doi = "10.1039/c5ib00013k", issn = "1757-9694", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150608-080917430", note = "© 2015 The Royal Society of Chemistry. \n\nReceived 17th January 2015, Accepted 19th May 2015, First published online 19 May 2015. \n\nWe thank Scott Fraser for providing the transfected fibroblasts. We thank the Biological Imaging Center of the Beckman Institute at Caltech for use of the two-photon microscope. \n\nThis work was funded by grants from the National Science Foundation (Division of Materials Research 0520565 and 1206121) and from the California Institute for Regenerative Medicine (RB5-07398). J.N. was supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1144469. A.L. was supported in part by a Rothschild Foundation fellowship. We thank Samuel A. Safran and Xinpeng Xu for reviewing the paper and providing useful comments.", revision_no = "23", abstract = "During processes such as development and cancer metastasis, cells migrate into three-dimensional fibrous matrices. Previous studies have speculated on the mechanical forces required for migration by observing matrix fiber alignment, densification, and degradation, but these forces remain difficult to quantify. Here we present a new experimental technique to simultaneously measure full-field 3D displacements and structural remodeling of a fibrous matrix, both of which result from cellular forces. We apply this “2-in-1” experimental technique to follow single cells as they invade a physiologically relevant fibrin matrix. We find that cells generate tube-like structures in the matrix by plastically deforming their surroundings, and they re-use these tubes to extend protrusions. Cells generate these tubular structures by applying both pulling and pushing forces.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/60976, title ="Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications", author = "Kulkarni, Chethana and Lo, Megan", journal = "Bioconjugate Chemistry", volume = "26", number = "10", pages = "2153-2160", month = "October", year = "2015", doi = "10.1021/acs.bioconjchem.5b00449", issn = "1043-1802", url = "https://resolver.caltech.edu/CaltechAUTHORS:20151012-111447715", note = "© 2015 American Chemical Society. \n\nReceived: August 10, 2015; Revised: September 11, 2015; Publication Date (Web): October 2, 2015. \n\nWe thank members of D.A.T.’s and T.K.U.’s research groups for helpful discussions. We are grateful to Prof. Stephen Mayo (Caltech) and Prof. Richard Kahn (Emory University) for their gifts of plasmids. The staff of the Proteome Exploration Laboratory of the Caltech Beckman Institute kindly assisted with mass spectrometry. Support for this work was provided by the Caltech Innovation Initiative (CI2), the Jacobs Institute for Molecular Engineering for Medicine, a Caltech Summer Undergraduate Research Fellowship (to M.L.), the Purdue Research Foundation, and a Purdue HHMI Summer Research Fellowship (to J.G.F.). \n\nThe authors declare no competing financial interest.", revision_no = "36", abstract = "Calmodulin (CaM) is a widely studied Ca^(2+)-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca^(2+) or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/59767, title ="Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro", author = "Ghazalli, Nadiah and Mahdavi, Alborz", journal = "Stem Cells and Development", volume = "24", number = "17", pages = "1995-2008", month = "August", year = "2015", doi = "10.1089/scd.2015.0007", issn = "1547-3287", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150819-105004630", note = "© 2015 Mary Ann Liebert, Inc. \n\nReceived for publication January 6, 2015, Accepted after revision May 4, 2015, Online Ahead of Editing: May 5, 2015, Online Ahead of Print: June 9, 2015, Published in Volume: 24 Issue 17: August 13, 2015. \n\nThis work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K.; U01DK089533 to A.D.R; and California Institute for Regenerative Medicine (CIRM) grant RB5-07398 to D.A.T. N.G. is supported by a CIRM predoctoral fellowship as part of an institutional grant to City of Hope. We thank the Light Microscopy Core, Electron Microscopy Core, and the Analytical Cytometry Core at City of Hope for providing technical assistance. \n\nThe authors declare no conflicts of interest.", revision_no = "27", abstract = "Postnatal pancreas is a potential source for progenitor cells to generate endocrine β-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for β-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional β-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133^(high) fraction and among 230 micro-manipulated single CD133^(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of β-like cells in vitro.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/60017, title ="Associative protein hydrogels", author = "Tirrell, David A. and Rapp, Peter B.", pages = "POLY-386", month = "August", year = "2015", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150901-153359534", note = "© 2015 American Chemical Society.", revision_no = "11", abstract = "Brad Olsen has made important contributions to our understanding of associative protein hydrogels. This lecture will\ndescribe some complementary efforts in our own lab., in which we are trying to det. the extent to which one can program the\nmacroscopic properties of mol. networks through the design and expression of artificial genes.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/60243, title ="Engineering energy dissipation in protein gels", author = "Dooling, Lawrence and Tirrell, David A.", pages = "POLY-191", month = "August", year = "2015", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150915-084509873", note = "© 2015 American Chemical Society.", revision_no = "9", abstract = "Protein gels are of interest as subjects of fundamental study and as matrixes for cell transplantation. But they\nare not very tough. This lecture will describe some recent expts. directed toward understanding and\nprogramming energy dissipation in artificial protein hydrogels.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/60030, title ="Methods for diversifying protein structure", author = "Tirrell, David A.", pages = "POLY-417", month = "August", year = "2015", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150902-104957229", note = "© 2015 American Chemical Society.", revision_no = "10", abstract = "By designing and expressing artificial genes, polymer chemists can prep. macromol. systems characterized by\nuniform chain length, sequence and stereochem. But the same mol. mechanisms that provide good control of\nchain architecture also limit the diversity of protein structure. This lecture will examine several\ncomplementary strategies for diversifying the products of cellular protein synthesis, including the\nincorporation of non-canonical amino acids, the prepn. of non-linear proteins, and programmed posttranslational\nmodification.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/60244, title ="Polymer chemist's perspective on protein science and engineering", author = "Tirrell, David A.", pages = "PMSE-198", month = "August", year = "2015", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150915-084643501", note = "© 2015 American Chemical Society.", revision_no = "9", abstract = "Synthetic polymers and proteins share the essential characteristic of long-chain mol. architecture. But they\ndiffer in important ways; proteins are uniform, often well folded, and evolvable, whereas polymers are\nheterogeneous and adopt random-coil or partially ordered conformations. This lecture will describe an ongoing\nattempt to bridge the gap between polymers and proteins by using artificial genes to direct the synthesis of\nartificial proteins, and by designing new amino acids that can be used in cellular protein synthesis. These\ndevelopments have provided a basis for new approaches to macromol. design, biol. imaging, and mol.-level\nanal. of cellular processes.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/59771, title ="Microbuckling of fibrin provides a mechanism for cell mechanosensing", author = "Notbohm, Jacob and Lesman, Ayelet", journal = "Journal of the Royal Society Interface", volume = "12", number = "108", pages = "Art. No. 20150320", month = "July", year = "2015", doi = "10.1098/rsif.2015.0320", issn = "1742-5689", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150819-141650427", note = "© 2015 The Author(s). Published by the Royal Society. \n\nReceived April 10, 2015. Accepted May 12, 2015. \n\nhis work was funded in part by a grant from the National Science Foundation (Division of Materials Research No. 0520565) through the Center for the Science and Engineering of Materials at the California Institute of Technology, and in part, by National Science Foundation grant no. DMR-1206121. J.N. was supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE-1144469. \n\nAuthors' contributions: J.N. and A.L. performed the experiments. J.N. performed the simulations. J.N. and P.R. wrote the manuscript. All authors discussed the results and gave approval for publication. \n\nWe have no competing interests.", revision_no = "22", abstract = "Biological cells sense and respond to mechanical forces, but how such a mechanosensing process takes place in a nonlinear inhomogeneous fibrous matrix remains unknown. We show that cells in a fibrous matrix induce deformation fields that propagate over a longer range than predicted by linear elasticity. Synthetic, linear elastic hydrogels used in many mechanotransduction studies fail to capture this effect. We develop a nonlinear microstructural finite-element model for a fibre network to simulate localized deformations induced by cells. The model captures measured cell-induced matrix displacements from experiments and identifies an important mechanism for long-range cell mechanosensing: loss of compression stiffness owing to microbuckling of individual fibres. We show evidence that cells sense each other through the formation of localized intercellular bands of tensile deformations caused by this mechanism.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/55416, title ="A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels", author = "DeForest, Cole A. and Tirrell, David A.", journal = "Nature Materials", volume = "14", number = "5", pages = "523-531", month = "May", year = "2015", doi = "10.1038/nmat4219", issn = "1476-1122", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150302-125620865", note = "© 2015 Macmillan Publishers Limited.\n\nReceived 15 July 2014; Accepted 15 January 2015; Published online 23 February 2015.\n\nThe authors thank P. Rapp for discussions on FRAP and FEM analysis, as well as for his constructive comments on the manuscript; M. Shahgholi and N. Torian for assistance with HRMS; J. Heath, J. Pfeilsticker and R. Henning for advice on peptide work and for use of their peptide synthesizer and HPLC; D. Koos and the Caltech Biological Imaging Center for use of confocal microscopes; and K. Beres and C. Murry for assistance with all Notch-related studies. This work was supported by the National Science Foundation Grant NSF-DMR 1206121 and a University of Washington Faculty Startup Grant (C.A.D.). \n\nContributions: C.A.D. and D.A.T. designed the experiments. C.A.D. conducted the experiments. C.A.D. and D.A.T. interpreted the data and composed the manuscript. \n\nAdditional information: Supplementary information is available in the online version of the paper. \n\nCompeting financial interests: The authors declare no competing financial interests.", revision_no = "29", abstract = "Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection–oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein \u200bvitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/54465, title ="Direct visualization of newly synthesized target proteins in situ", author = "tom Dieck, Susanne and Kochen, Lisa", journal = "Nature Methods", volume = "12", number = "5", pages = "411-414", month = "March", year = "2015", doi = "10.1038/nmeth.3319", issn = "1548-7091", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150205-174152503", note = "© 2015 Macmillan Publishers Limited. \n\nReceived 28 April 2014; Accepted 29 January 2015; Published online 16 March 2015. \n\nWe thank N. Fuerst and A. Staab for the preparation of cultured hippocampal neurons. E.M.S. is funded by the Max Planck Society; an Advanced Investigator award from the European Research Council, Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center (CRC) 902: Molecular Principles of RNA-based Regulation; DFG CRC 1080: Molecular and Cellular Mechanisms of Neural Homeostasis; and the DFG Cluster of Excellence for Macromolecular Complexes, Goethe University, Frankfurt. D.A.T is funded by US National Institutes of Health grant R01 GM062523.", revision_no = "95", abstract = "Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/54160, title ="Cell-Specific Proteomic Analysis in Caenorhabditis elegans", author = "Yuet, Kai P. and Doma, Meenakshi K.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "112", number = "9", pages = "2705-2710", month = "March", year = "2015", doi = "10.1073/pnas.1421567112", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150127-223214858", note = "© 2015 National Academy of Sciences.\n\nEdited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved January 22, 2015 (received for review November 10, 2014).\nPublished online before print February 17, 2015.\n\nWe thank past and present members of the P.W.S.\nand D.A.T. laboratories for fruitful discussions and suggestions, and Roxana\nEggleston-Rangel and Geoffrey T. Smith (Proteome Exploration Laboratory,\nBeckman Institute, California Institute of Technology) for technical assistance.\nThis work was supported by National Institutes of Health (NIH) Grant\nR01 GM062523. K.P.Y. was supported in part by a National Science Foundation\ngraduate fellowship. P.W.S. is an Investigator of the Howard Hughes\nMedical Institute, which supported this work. The Proteome Exploration\nLaboratory is supported by Gordon and Betty Moore Foundation Grant\nGBMF775, the Beckman Institute, and NIH Grant 1S10RR029594-01A1.\n\nAuthor contributions: K.P.Y., M.K.D., J.T.N., E.M.S., P.W.S., and D.A.T. designed research;\nK.P.Y., M.K.D., and J.T.N. performed research; M.J.S., R.L.J.G., A.M., and S.H. contributed\nnew reagents/analytic tools; K.P.Y., M.J.S., P.W.S., and D.A.T. analyzed data; and K.P.Y.,\nP.W.S., and D.A.T. wrote the paper.\n\nThe authors declare no conflict of interest.\n\nThis article is a PNAS Direct Submission.\n\nData deposition: The vectors generated in this study have been deposited in the Addgene\ndatabase, www.addgene.org (Addgene nos. 62598 and 62599).\n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1421567112/-/DCSupplemental.", revision_no = "27", abstract = "Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53888, title ="A Qrr Noncoding RNA Deploys Four Different Regulatory Mechanisms to Optimize Quorum-Sensing Dynamics", author = "Feng, Lihui and Rutherford, Steven T.", journal = "Cell", volume = "160", number = "1-2", pages = "228-240", month = "January", year = "2015", doi = "10.1016/j.cell.2014.11.051", issn = "0092-8674", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-132939806", note = "© 2015 Elsevier Under an Elsevier user license. Received 2 May 2014, Revised 20 September 2014, Accepted 14 November 2014, Available online 8 January 2015.\n\nWe thank Terence Hwa for generously providing the BW-RI strain and the pZA31-lucNB and pZE12G plasmids. We are indebted to members of the B.L.B. and N.S.W. laboratories for insightful discussions and suggestions. This work was supported by the Howard Hughes Medical Institute, NIH grant 5R01GM065859 and National Science Foundation (NSF) grant MCB-0343821 to B.L.B., NIH grant R01GM082938 to N.S.W., and NIH grant R01GM062523 and the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office to D.A.T. K.P. is supported by a postdoctoral fellowship from the Human Frontiers in Science Program (HFSP). S.T.R. was supported by NIH fellowship F32AI085922. J.C.V.K. is supported by funds from Indiana University.", revision_no = "25", abstract = "Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/63368, title ="Cell culture and cell based sensor on CMOS", author = "Wang, Hua and Mahdavi, Alborz", pages = "468-471", month = "October", year = "2014", doi = "10.1109/BioCAS.2014.6981764", isbn = "978-1-4799-2346-5", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160105-075920380", note = "© 2014 IEEE.", revision_no = "12", abstract = "This paper focuses on CMOS biosensor technologies for cellular biosensing applications. We first present our technologies to achieve on-CMOS cell culture, maintenance, and differentiation, as the basis for CMOS cellular biosensors. Next, we introduce a CMOS frequency-shift magnetic sensor scheme which performs detections without post-processing or external biasing magnetic field. Finally, we will demonstrate a CMOS magnetic cell based sensor which achieves real-time chemical detections; such a sensor scheme can be utilized for massively paralleled high-throughput chemical screening in drug development.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47532, title ="Cell Surface Display Yields Evolvable, Clickable Antibody Fragments", author = "Van Deventer, James A. and Yuet, Kai P.", journal = "ChemBioChem", volume = "15", number = "12", pages = "1777-1781", month = "August", year = "2014", doi = "10.1002/cbic.201402184", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140728-141903775", note = "© 2014 Wiley-VCH Verlag GmbH & Co. \n\nArticle first published online: 17 Jul 2014.\n\nWe thank Mona Shahgholi for MALDI mass spectrometry assistance at the Caltech Chemistry Mass Spectrometry facility, Igor\nAntoshechkin and the Millard and Muriel Jacobs Genetics and\nGenomics Laboratory for assistance with high-throughput sequencing, Cynthia Shuman of GE Healthcare for Biacore kinetic assay guidance, Janek Szychowski and Alborz Mahdavi for materials, and Maren Buck for helpful comments on the manuscript. This work was supported by NIH grant R01 GM62523. J.A.V. was supported in part by a National Defense Science and Engineering Graduate (NDSEG) fellowship, and K.P.Y. in part by a National Science Foundation (NSF) Graduate Fellowship.", revision_no = "37", abstract = "Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide–alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/47388, title ="Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry", author = "Sun, Fei and Zhang, Wen-Bin", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "111", number = "31", pages = "11269-11274", month = "August", year = "2014", doi = "10.1073/pnas.1401291111", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140722-093927552", note = "Copyright © 2014 National Academy of Sciences. \n\nEdited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved July 1, 2014 (received for review January 21, 2014). Published ahead of print July 21, 2014. \n\nThis research was funded by National Science Foundation Grant DMR 1206121 and by the Gordon and Betty Moore Foundation through Grant GBMF2809 to the California Institute of Technology Programmable Molecular Technology Initiative. \n\nF.S. and W.-B.Z. contributed equally to this work. \n\nAuthor contributions: F.S., W.-B.Z., F.H.A., and D.A.T. designed research; F.S., W.-B.Z., and A.M. performed research; F.S., W.-B.Z., and A.M. contributed new reagents/analytic tools; F.S., W.-B.Z., A.M., F.H.A., and D.A.T. analyzed data; and F.S., W.-B.Z., A.M., F.H.A., and D.A.T. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1401291111/-/DCSupplemental.", revision_no = "22", abstract = "Protein-based hydrogels have emerged as promising alternatives to synthetic hydrogels for biomedical applications, owing to the precise control of structure and function enabled by protein engineering. Nevertheless, strategies for assembling 3D molecular networks that carry the biological information encoded in full-length proteins remain underdeveloped. Here we present a robust protein gelation strategy based on a pair of genetically encoded reactive partners, SpyTag and SpyCatcher, that spontaneously form covalent isopeptide linkages under physiological conditions. The resulting “network of Spies” may be designed to include cell-adhesion ligands, matrix metalloproteinase-1 cleavage sites, and full-length globular proteins [mCherry and leukemia inhibitory factor (LIF)]. The LIF network was used to encapsulate mouse embryonic stem cells; the encapsulated cells remained pluripotent in the absence of added LIF. These results illustrate a versatile strategy for the creation of information-rich biomaterials.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/48310, title ="Dynamic biomacromolecular patterning of photoresponsive hydrogels", author = "Tirrell, David A. and DeForest, Cole A.", pages = "PMSE 183", month = "August", year = "2014", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140811-130624245", note = "© 2014 American Chemical Society.", revision_no = "10", abstract = "Polymer-based hydrogels have emerged as a unique class of biomaterials that enable stem cells to be cultured in three-dimensions within near-physiol., synthetic microenvironments. Recent strategies have been developed that permit bioepitopes (e.g., peptides, full-length proteins) to be introduced at any point in time and space to affect cell function spatiotemporally within user-defined subvolumes of the bulk material. While these techniques have been successfully utilized to direct a variety of basic cellular functions, advanced platforms that permit biol. cues to be both introduced and subsequently removed would be beneficial in recapitulating the dynamic abundance of signaling biomols. in the native, temporally-variable niche and in modulating complex cellular behavior. In this work, we demonstrate that the combination of two bioorthogonal light-based chemistries provides for the reversible immobilization of protein cues spatially within a hydrogel. The highlighted approach enables precise control over 4D biochem. functionalization of a synthetic polymer network in response to user-defined photonic stimuli. Results further highlight the versatility of such dynamic biomacromol. signal presentation in better understanding basic cell physiol.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44082, title ="In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry", author = "Hatzenpichler, Roland and Scheller, Silvan", journal = "Environmental Microbiology", volume = "16", number = "8", pages = "2568-2590", month = "August", year = "2014", doi = "10.1111/1462-2920.12436", issn = "1462-2912", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140303-103306009", note = "© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.\nThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.\n\nAccepted manuscript online: 26 Feb 2014; Manuscript Accepted: 18 Feb 2014; Manuscript Received: 20 Dec 2013. \n\nThis article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1462-2920.12436. \n\nWe thank Katherine Dawson and Hiroyuki Imachi for advice on anaerobic culturing, Yunbin Guan for assistance with nanoSIMS analyses, John D. Bagert for discussions on click chemistry, Grayson Chadwick for calculating genomic Met contents, and Jennifer Glass for helpful comments on an early version of this manuscript. We acknowledge the Caltech Proteome Exploration Laboratory (PEL) staff for analyzing mass spectrometry samples and their technical assistance with sample preparation and interpretation of results. The PEL is supported by the Beckman Institute and the Gordon & Betty Moore Foundation. Roland Hatzenpichler was supported via an O.K. Earl Postdoctoral Scholarship awarded by Caltech’s Division of Geological and Planetary Sciences as well as an Erwin Schrӧdinger Postdoctoral Fellowship of the Austrian Science Fund (FWF), J 3162-B20. Silvan Scheller was supported by the Swiss National Science Foundation (grant PBEZP2_142903). Funding for this project was provided by the Gordon and Betty Moore Foundation through Grant GBMF3780 to VJO, grant from the Department of Energy (DE-PS02-09ER09-25) to VJO, and by a National Institutes of Health grant NIH R01 GM062523 to DAT.\n\nPlease note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.", revision_no = "58", abstract = "Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for L-methionine, followed by fluorescent labeling of AHA containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater, and anoxic sediment. We also developed combined assays that couple BONCAT with rRNA-targeted FISH, enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labeling by nanoSIMS (^(15)NH_4^+ assimilation) for individual cells within a sediment sourced enrichment culture showed concordance between AHA positive cells and ^(15)N enrichment. BONCAT-FISH offers a fast, inexpensive, and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single cell level.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/48607, title ="Time-\u200bresolved, cell-\u200bselective analysis of protein synthesis in complex biological systems", author = "Tirrell, David A.", pages = "BIOL 148", month = "August", year = "2014", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140815-103023347", note = "© 2014 American Chemical Society.", revision_no = "8", abstract = "The behavior of biol. systems varies in space and time. The BONCAT (bioorthogonal non-canonical amino acid tagging) method provides a means by which investigators can probe the heterogeneous nature of protein synthesis in complex biol. systems, including live animals. This lecture will examine the scope and limitations of the BONCAT method, with examples drawn from neurobiol., cancer biol. and microbiol.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44117, title ="Quantitative, Time-Resolved Proteomic Analysis by Combining Bioorthogonal Noncanonical Amino Acid Tagging and Pulsed Stable Isotope Labeling by Amino Acids in Cell Culture", author = "Bagert, John D. and Xie, Yushu J.", journal = "Molecular and Cellular Proteomics", volume = "13", number = "5", pages = "1352-1358", month = "May", year = "2014", doi = "10.1074/mcp.M113.031914", issn = "1535-9476", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140304-081227131", note = "© 2014 American Society for Biochemistry and Molecular Biology, Inc. \n\nFirst Published on February 21, 2014. Received June 18, 2013, and in revised form, January 9, 2014 Published, MCP Papers in Press, February 21, 2014, DOI 10.1074/mcp.M113.031914. \n\nThis work was supported by National Institutes of Health grant NIH RO1 GM062523, the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from U.S. Army Research Office, the Joseph J. Jacobs Institute for Molecular Engineering for Medicine, the Betty and Gordon Moore Foundation through Grant GBMF775, and the Beckman Institute. Y.J.X. acknowledges funding from the Caltech Summer Undergraduate Research Fellowships (SURF) program. We thank Kai Yuet for providing the E. coli KY2 strain and the PEL staff for technical support.", revision_no = "51", abstract = "An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30-min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/46067, title ="Contractile forces regulate cell division in three-dimensional environments", author = "Lesman, Ayelet and Notbohm, Jacob", journal = "Journal of Cell Biology", volume = "205", number = "2", pages = "155-162", month = "April", year = "2014", doi = "10.1083/jcb.201309029", issn = "0021-9525", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140604-075231845", note = "© 2014 Lesman et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication\ndate (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Submitted: 6 September 2013\nAccepted: 25 March 2014. We thank Scott Fraser for providing the transfected fibroblasts. We thank the Biological Imaging Center at Caltech for use of the two-photon microscope. This research was supported by grants from the National Science Foundation (Division of Materials Research No. 0520565 and 1206121). J. Notbohm was supported by the Department of Defense through the National Defense Science and Engineering Graduate Fellowship Program. A. Lesman was supported in part by the Rothschild fellowship foundation.\nThe authors declare no competing financial interests.", revision_no = "44", abstract = "Physical forces direct the orientation of the cell division axis for cells cultured on rigid, two-dimensional (2D) substrates. The extent to which physical forces regulate cell division in three-dimensional (3D) environments is not known. Here, we combine live-cell imaging with digital volume correlation to map 3D matrix displacements and identify sites at which cells apply contractile force to the matrix as they divide. Dividing cells embedded in fibrous matrices remained anchored to the matrix by long, thin protrusions. During cell rounding, the cells released adhesive contacts near the cell body while applying tensile forces at the tips of the protrusions to direct the orientation of the cell division axis. After cytokinesis, the daughter cells respread into matrix voids and invaded the matrix while maintaining traction forces at the tips of persistent and newly formed protrusions. Mechanical interactions between cells and the extracellular matrix constitute an important mechanism for regulation of cell division in 3D environments.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/45637, title ="Brush-first and Click: Efficient Synthesis of Nanoparticles that Degrade and Release Doxorubicin in Response to Light", author = "Burts, Alan O. and Liao, Longyan", journal = "Photochemistry and Photobiology", volume = "90", number = "2", pages = "380-385", month = "March", year = "2014", doi = "10.1111/php.12182 ", issn = "0031-8655", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140509-095521349", note = "© 2013 The American Society of Photobiology.\n\nReceived 14 August 2013, accepted 30 September 2013.\n\nArticle first published online: 25 Nov. 2013.\n\nThis work has been supported in part by the MIT\nDepartment of Chemistry, the MIT Research Support Committee, and the\nMIT Lincoln Laboratories Advanced Concepts Committee.", revision_no = "11", abstract = "New strategies for the synthesis of multifunctional particles that respond to external stimuli and release biologically relevant agents will enable the discovery of new formulations for drug delivery. In this article, we combine two powerful methods: brush-first ring-opening metathesis polymerization and copper-catalyzed azide–alkyne cycloaddition click chemistry, for the synthesis of a novel class of brush-arm star polymers (BASPs) that simultaneously degrade and release the anticancer drug doxorubicin (DOX) in response to 365 nm light. In vitro cell viability studies were performed to study the toxicity of azide- and DOX-loaded BASPs. The former were completely nontoxic. The latter showed minimal toxicity in the absence of light; UV-triggered DOX release led to IC_(50) values that were similar to that of free DOX.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/44658, title ="Chemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics", author = "Yuet, Kai P. and Tirrell, David A.", journal = "Annals of Biomedical Engineering", volume = "42", number = "2", pages = "299-311", month = "February", year = "2014", doi = "10.1007/s10439-013-0878-3", issn = "0090-6964", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140404-094604296", note = "© 2013 Biomedical Engineering Society.\n\nReceived 11 July 2013; accepted 24 July 2013; published online 14 August 2013.\n\nWork at Caltech on non-canonical amino acid tagging is supported by National Institutes of Health grant NIH R01 GM062523 and by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from U.S. Army Research Office.", revision_no = "21", abstract = "Accurate measurements of the abundances, synthesis rates and degradation rates of cellular proteins are critical for understanding how cells and organisms respond to changes in their environments. Over the past two decades, there has been increasing interest in the use of mass spectrometry for proteomic analysis. In many systems, however, protein diversity as well as cell and tissue heterogeneity limit the usefulness of mass spectrometry-based proteomics. As a result, researchers have had difficulty in systematically identifying proteins expressed within specified time intervals, or low abundance proteins expressed in specific tissues or in a few cells in complex microbial systems. In this review, we present recently-developed tools and strategies that probe these two subsets of the proteome: proteins synthesized during well-defined time intervals—temporally resolved proteomics—and proteins expressed in predetermined cell types, cells or cellular compartments—spatially resolved proteomics—with a focus on chemical and biological mass spectrometry-based methodologies.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/42825, title ="Prometastatic GPCR CD97 Is a Direct Target of Tumor Suppressor microRNA-126", author = "Lu, Ying Y. and Sweredoski, Michael J.", journal = "ACS Chemical Biology", volume = "9", number = "2", pages = "334-338", month = "February", year = "2014", doi = "10.1021/cb400704n", issn = "1554-8929", url = "https://resolver.caltech.edu/CaltechAUTHORS:20131204-105211879", note = "© 2013 American Chemical Society. \n\nReceived: September 12, 2013; Accepted: November 12, 2013. Publication Date (Web): November 25, 2013. \n\nWe thank R. Graham, A. Moradian, and G. Smith at the\nProteome Exploration Laboratory of the Beckman Institute at\nCaltech for assistance with proteomic studies. We thank K.\nFang, K. Yuet, and L. Dooling for cloning advice and R.\nDiamond for assistance with flow cytometry. This work was\nsupported by National Institutes of Health grant NIH R01\nGM062523. The Proteome Exploration Laboratory is supported\nby the Caltech Beckman Institute and by the Gordon\nand Betty Moore Foundation through Grant GBMF775.", revision_no = "30", abstract = "Tumor suppressor microRNA-126 (miR-126) is often down-regulated in cancer cells, and its overexpression is found to inhibit cancer metastasis. To elucidate the mechanism of tumor suppression by miR-126, we analyzed the proteomic response to miR-126 overexpression in the human metastatic breast cancer cell line MDA-MB-231. To acquire quantitative, time-resolved information, we combined two complementary proteomic methods, BONCAT and SILAC. We discovered a new direct target of miR-126: CD97, a pro-metastatic G-protein-coupled receptor (GPCR) that has been reported to promote tumor cell invasion, endothelial cell migration, and tumor angiogenesis. This discovery establishes a link between down-regulation of miR-126 and overexpression of CD97 in cancer and provides new mechanistic insight into the role of miR-126 in inhibiting both cell-autonomous and non-cell-autonomous cancer progression.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/43163, title ="Identification of secreted bacterial proteins by noncanonical amino acid tagging", author = "Mahdavi, Alborz and Szychowski, Janek", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "111", number = "1", pages = "433-438", month = "January", year = "2014", doi = "10.1073/pnas.1301740111", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20131224-093455338", note = "© 2014 National Academy of Sciences. \n\nEdited by Ralph R. Isberg, Howard Hughes Medical Institute, Tufts University School of Medicine, Boston, MA, and approved November 25, 2013 (received for review January 27, 2013). \n\nWe thank Geoff Smith (Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology) for technical assistance. This work was supported by the National Institutes of Health (Grant R01 GM062523), the Institute for Collaborative Biotechnologies (Grant W911NF-09-0001 from the US Army Research Office), the Burroughs\nWellcome Fund in the Pathogenesis of Infectious Disease, and the Gordon and Betty Moore Foundation. A.M. is the recipient of a Natural Sciences and Engineering Reasearch Council of Canada scholarship and a Donna and Benjamin M. Rosen postgraduate scholarship.", revision_no = "33", abstract = "Microbial pathogens use complex secretion systems to deliver virulence factors into host cells, where they disrupt host cell function. Understanding these systems is essential to the development of new treatments for infectious disease. A challenge in such studies arises from the abundance of host cell proteins, which interfere with detection of microbial effectors. Here we describe a metabolic labeling strategy that allows selective enrichment of microbial proteins from the host cell cytoplasm. The method enables efficient identification of microbial proteins that have been delivered to the host, identifies distinct secretion profiles for intracellular and extracellular bacteria, and allows for determination of the order of injection of microbial proteins into host cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/52627, title ="Colony-Forming Progenitor Cells in the Postnatal Mouse Liver and Pancreas Give Rise to Morphologically Distinct Insulin-Expressing Colonies in 3D Cultures", author = "Jin, Liang and Feng, Tao", journal = "Review of Diabetic Studies", volume = "11", number = "1", pages = "35-50", month = "January", year = "2014", doi = "10.1900/RDS.2014.11.35", issn = "1613-6071", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141222-133544740", note = "Copyright © by Lab & Life Press/SBDR \n\nManuscript submitted February 15, 2013; resubmitted June 20, 2013; accepted July 9, 2013. \n\nWe thank Lucy Brown and Alexander Spalla from the Analytical Cytometry Core of City of Hope for assistance in flow sorting. This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., U01DK089533 to A.D.R., National Science Foundation NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T., and NIH P30CA33572 to Analytical Cytometry Core at City of Hope. We also gratefully acknowledge support from Ella Fitzgerald Charitable Foundation, John C. Hench Foundation, Gordon Ross Medical Foundation, and Oxnard Foundation. \n\nThis work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., U01DK089533 to A.D.R., and P30 CA33572 to the Analytical Cytometry Core at City of Hope, and by National Science Foundation grant DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T.", revision_no = "19", abstract = "In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed “Dark” colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency\n(~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133^(+)CD49f^(low)CD107b^(low) phenotype, while pancreatic CFU-Dark are CD133^-. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/41424, title ="Selective Functionalization of the Protein N Terminus with N-Myristoyl Transferase for Bioconjugation in Cell Lysate", author = "Kulkarni, Chethana and Kinzer-Ursem, Tamara L.", journal = "ChemBioChem", volume = "14", number = "15", pages = "1958-1962", month = "October", year = "2013", doi = "10.1002/cbic.201300453", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130919-145751587", note = "© 2013 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim.\nReceived: July 11, 2013.\n\nArticle first published online: 12 Sep. 2013.\n\nWe thank John Ngo, Rebecca Connor, David Ralin, Sandy Tungteakkhun,\nand Claudia Lee for helpful discussions, and Mona\nShahgoli and Robert Graham for assistance with mass spectrometry\nstudies. Support for this work was provided by the Jacobs\nInstitute for Molecular Engineering for Medicine and the Caltech\nInnovation Initiative.", revision_no = "16", abstract = "A site to behold: Robust site-specific functionalization of engineered proteins is achieved with N-myristoyl transferase (NMT) in bacterial cells. NMT tolerates non-natural substrate proteins as well as reactive fatty acid tags, rendering it a powerful tool for protein conjugation applications, including the construction of protein microarrays from lysate.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/43920, title ="Controlling Macromolecular Topology with Genetically Encoded SpyTag-SpyCatcher Chemistry", author = "Zhang, Wen-Bin and Sun, Fei", journal = "Journal of the American Chemical Society", volume = "135", number = "37", pages = "13988-13997", month = "September", year = "2013", doi = "10.1021/ja4076452 ", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140221-084933853", note = "© 2013 American Chemical Society.\n\nReceived: July 24, 2013; Published: August 21, 2013.\n\nThis research was funded by National Science Foundation (DMR 1206121) and by the Gordon and Betty Moore\nFoundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative.", revision_no = "15", abstract = "Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag–SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag–SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag–SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.\n", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/41849, title ="Cellular synthesis of non-linear proteins", author = "Zhang, Wen-Bin and Sun, Fei", pages = "POLY-4", month = "September", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20131010-083215575", note = "© 2013 American Chemical Society.", revision_no = "14", abstract = "The spontaneous isopeptide bond formation between a peptide tag (SpyTag) and its protein partner (SpyCatcher) allows the\nsynthesis of non-linear proteins in cellular hosts. Strategic placement of sequences encoding SpyTag and SpyCatcher\nwithin protein-coding genes programs the post-translational modification of the expressed proteins in situ, and enables the\nsynthesis of a variety of unconventional protein topologies. For example, when SpyTag and SpyCatcher were placed at the Nand\nC-termini, resp., of an elastin-like-protein (ELP), highly efficient cyclization of the ELP was obsd. At low protein synthesis\nrates (e.g., at low temp. in relatively poor media), the product was almost exclusively monomeric circular ELP; at high protein\nsynthesis rates (e.g., at high temp. in rich media), the major product was monomeric circular protein while significant amts. of\nchain-extended oligomers were also obsd. The circular topol. has been demonstrated by SDS-PAGE, MALDI-TOF mass\nspectrometry, proteolytic digestion, and single-site mutation.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/41934, title ="Molecular and cellular recognition in engineering protein hydrogels", author = "Tirrell, David A.", pages = "PMSE-53", month = "September", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20131016-082304830", note = "© 2013 American Chemical Society.", revision_no = "11", abstract = "This lecture will explore strategies for the use of mol. recognition in the design of protein hydrogels. Control\nof gel structure, dynamics and phys. properties will be examd., and opportunities for control of cellular behavior\nwill be described.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/39260, title ="Strain propagation in artificial extracellular matrix proteins can accelerate cell spreading and polarization", author = "Tzlil, Shelly and Tirrell, David A.", journal = "Soft Matter", volume = "9", number = "23", pages = "5602-5608", month = "May", year = "2013", doi = "10.1039/c3sm27137d", issn = "1744-6848", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130709-083629634", note = "© 2013 The Royal Society of Chemistry.\n\nReceived 15th September 2012.\nAccepted 29th April 2013.\nFirst published online 07 May 2013.\n\nWe acknowledge Dr Eileen Fong for discussion and help in\nprotein expression. We also thank Dr Zhen-Gang Wang for\nadvice on the simulation model. S.T. was supported by the\nHuman Frontier Science Program Cross-Disciplinary Fellowship.\nThis work was supported by the NSF Center for the Science\nand Engineering of Materials and by NSF DMR 1206121 at the\nCalifornia Institute of Technology.", revision_no = "16", abstract = "In recent years the ability of cells to sense the mechanical properties of their environments and deform them locally has become increasingly clear. To better understand the mechanical coupling between cells and their surroundings, we have examined the dynamics of adhesion of Chinese hamster ovary cells cultured on engineered protein substrates with different viscoelastic properties. We find that cell spreading and polarization rates vary two- and five-fold, respectively, for crosslinked and uncrosslinked proteins, despite the fact that the rates of growth of individual adhesion complexes on the different substrates are comparable. A wave of adhesion growth along the cell contour is observed by total internal reflection fluorescence microscopy for cells plated on crosslinked materials, but not on uncrosslinked substrates. We propose a mechanism in which cell-induced strains accumulate in crosslinked materials as a result of adhesion growth. Strain propagation within the material explains the observed adhesion growth patterns and the increased rates of spreading and polarization characteristic of cells cultured on crosslinked substrates. We investigate the proposed mechanism through Brownian dynamics simulation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38561, title ="Self-Assembly of Elastin–Mimetic Double Hydrophobic Polypeptides", author = "Le, Duc H. T. and Hanamura, Ryo", journal = "Biomacromolecules", volume = "14", number = "4", pages = "1028-1034", month = "April", year = "2013", doi = "10.1021/bm301887m", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130517-131452178", note = "© 2013 American Chemical Society.\n\nReceived: December 10, 2012; Revised: February 27, 2013;\nPublished: March 18, 2013.\n\nThis work was supported by a Grant-in-Aid for Scientific\nResearch (No. 22107005) on the Innovative Areas: “Fusion\nMaterials” (Area No. 2206) from the Ministry of Education,\nCulture, Sports, Science and Technology (MEXT), Japan.\nD.H.T.L. is grateful to a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science (JSPS). A part of this work was conducted at the Center for Nanobio\nIntegration (CNBI), Center for Medical System Integration\n(CMSI), and Center for Nano Lithography and Analysis, The\nUniversity of Tokyo, supported by MEXT. We thank Profs. Yukio Yamaguchi and Teruyuki Nagamune for their help in the\npreparation of polypeptides.", revision_no = "13", abstract = "We have constructed a novel class of “double-hydrophobic” block polypeptides based on the hydrophobic domains found in native elastin, an extracellular matrix protein responsible for the elasticity and resilience of tissues. The block polypeptides comprise proline-rich poly(VPGXG) and glycine-rich poly(VGGVG), both of which dehydrate at higher temperature but form distinct secondary structures, β-turn and β-sheet respectively. In water at 45 °C, the block polypeptides initially assemble into nanoparticles rich in β-turn structures, which further connect into long (>10 μm), beaded nanofibers along with the increase in the β-sheet content. The nanofibers obtained are well-dispersed in water, and show thermoresponsive properties. Polypeptides comprising each block component assemble into different morphologies, showing that the conjugation of poly(VPGXG) and poly(VGGVG) plays a role for beaded fiber formation. These results may provide innovative ideas for designing peptide-based materials but also opportunities for developing novel materials useful for tissue engineering and drug delivery systems.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37811, title ="Attaching proteins to polymers and other things", author = "Tirrell, David A.", pages = "POLY 102", month = "April", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130408-145117823", note = "© 2013 American Chemical Society.", revision_no = "18", abstract = "This lecture will discuss several new approaches to protein modification, including the use\nof non-canonical amino acids and the enzyme N-myristoyltransferase. Protein pegylation,\nsurface conjugation, and reversible attachment to polymer gels, will be described.\nApplications in protein therapeutics, microarray technol., and high-throughput biochem. will be explored.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/39723, title ="Cell type-specific proteomic profiling in Caenorhabditis elegans", author = "Yuet, Kai P. and Doma, Meenakshi K.", pages = "BIOL 243", month = "April", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130802-084724182", note = "© 2013 American Chemical Society.", revision_no = "15", abstract = "In a complex eukaryote like C. Elegans, cell and tissue heterogeneity restricts the usefulness of large-scale, mass spectrometrybased anal. of an organism's proteome. As worm cell lines are not available and enriching for specific cells or tissues is challenging, researchers cannot systematically identify low abundance proteins expressed in specific cells or tissues from wholeworm lysates. To isolate protein from specific cells, we have engineered C. Elegans phenylalanyl-tRNA synthetases capable of appending azide- or alkynyl-bearing analogs of phenylalanine to tRNA^(Phe). As these analogs are not substrates for any of the wild-type aminoacyl-tRNA synthetases in C. Elegans, we achieved cell type selectivity by spatially restricting the expression of a mutant synthetase using cell type-specific promoters. Because proteins from those cells that express the mutant synthetase - and only those cells - contain the analogs, they can be enriched by treatment with alkyne- or azide-functionalized biotin reagents and subsequent affinity chromatog. for identification by LC/MS-MS.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37851, title ="Engineering the mechanical properties of protein-based hydrogels prepared by thiol-maleimide chemistry", author = "Zhang, Wenbin and Tirrell, David A.", pages = "PMSE 234", month = "April", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130410-101809254", note = "© 2013 American Chemical Society", revision_no = "14", abstract = "Genetic engineering methods were applied to design and biosynthesis of telechelic\nproteins from elastin- and fibronectin-derived repeating units. The telechelic proteins\nbearing terminal thiols could either undergo chain-extension with bis-maleimidefunctionalized\npoly(ethylene glycol) (MAL-PEG-MAL) or crosslinking with tetrakismaleimide-\nfunctionalized 4-arm star PEG (star-PEG-MAL). The latter leads to proteinbased\nhydrogels that are transparent, uniform, and highly extensible. The gelation time\nranges from several minutes to a few hours depending on the free-thiol content, the\nprotein wt. percentage, and tris(2-carboxyethyl)phosphine concn. The mech. properties of\nthe gel depend on the protein content and the cross-linker concn. and can be further\ntuned by using a mixt. of MAL-PEG-MAL and star-PEG-MAL for crosslinking. The water\ncontents of the hydrogels are high, esp. after swelling. The results suggest its promising\napplication for cell encapsulation and 3D cell culture in tissue engineering.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/39767, title ="Non-canonical amino acids in the interrogation of cellular protein synthesis", author = "Tirrell, David A.", pages = "ORGN 353", month = "April", year = "2013", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130805-131101494", note = "© 2013 American Chemical Society.", revision_no = "11", abstract = "Proteins can be rendered susceptible to bio-orthogonal chemistries through metabolic labeling with appropriately designed, noncanonical\namino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces, in whole or in\npart, one of the natural amino acids specified by the protein gene(s) of interest. This approach allows the ncAA to be introduced\nat a controlled rate into positions normally occupied by the natural amino acid residue. Because this method permits labeling of\nproteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with\nreactive ncAAs. In procedures similar to those used in isotopic labeling, translationally active ncAAs are incorporated into\nproteins during a \"pulse\" in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from\nthose made prior to the pulse through bio-orthogonal ligation of the ncAA side chain to probes that permit detection, isolation,\nand visualization of the labeled proteins. The selectivity of the method can be enhanced through the use of mutant aminoacyltRNA\nsynthetases (aaRSs) that permit incorporation of ncAAs that are not used by the endogenous translational machinery.\nControlled expression of mutant synthetases has been combined with ncAA-tagging to permit cell-selective and state-selective\nmetabolic labeling of proteins. Expression of a mutant synthetase in a subset of cells in a complex cellular mixt. - or in a live\nanimal - restricts labeling to that subset; proteins synthesized in cells that do not express the synthetase are neither labeled nor\ndetected.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38775, title ="Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells", author = "Ngo, John T. and Schuman, Erin M.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "110", number = "13", pages = "4992-4997", month = "March", year = "2013", doi = "10.1073/pnas.1216375110 ", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130604-100505112", note = "© 2013 National Academy of Sciences.\n\nEdited by Paul Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved February 11, 2013 (received for review September 19, 2012).\n\nPublished online before print March 11, 2013.\n\nWe thank Michael Sweredowski, Robert Graham, and Sonja Hess of the Proteome Exploration Laboratory (Beckman Institute, Caltech) for their help and advice in collecting and analyzing tandem mass spectrometry data; Angela Ho and Jost Vielmetter of the Protein Expression Center (Beckman\nInstitute, Caltech) for their help in generating stable cell lines; Carolyn Bertozzi and her laboratory (University of California, Berkeley) for their gift of DIFOAlexa Fluor 488; and John Phillips (Caltech) and members of D.A.T.’s laboratory for helpful discussions. Support for this work was provided by National Institutes of Health Grant R01 GM062523, the Joseph J. Jacobs Institute for Molecular Engineering for Medicine, and the Institute for Collaborative Biotechnologies through Grant W911NF-09-0001 from the US Army Research Office.\n\nAuthor contributions: J.T.N. and D.A.T. designed research; J.T.N. performed research; J.T.N.,\nE.M.S., and D.A.T. analyzed data; and J.T.N., E.M.S., and D.A.T. wrote the paper.", revision_no = "22", abstract = "Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38078, title ="Colony-forming cells in the adult mouse pancreas are expandable in Matrigel and form endocrine/acinar colonies in laminin hydrogel", author = "Jin, Liang and Feng, Tao", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "110", number = "10", pages = "3907-3912", month = "March", year = "2013", doi = "10.1073/pnas.1301889110 ", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130423-105847349", note = "© 2013 National Academy of Sciences. \n\nFreely available online through the PNAS open access option. \n\nContributed by Arthur D. Riggs, January 29, 2013 (sent for review December 26, 2012). Published online before print February 19, 2013. \n\nWe thank Lucy Brown, Alexander Spalla, and Pavinder Kaur from the Analytical Cytometry Core of City of Hope for assistance in flow sorting and Dr. Vincenzo Cirulli at the University of Washington for evaluating photomicrographs of electron microscopy. This work is supported in part by National Institutes of Health (NIH) Grant R01DK081587 (to H.T.K.), U01DK089533 (to A.D.R.), DK078803 (to M.S.), National Science Foundation NSF-DMR-1206121 (to D.A.T.), Office of Naval Research ONR-N00014-02-1 0958 and NSF-DBI-9970143 (to Electron Microscopy Core), NIH Grant P30 CA33572 (to Analytical Cytometry Core at City of Hope), a Juvenile Diabetes Research Foundation postdoctoral fellowship (to H.P.S.), and a City of Hope Eugene and Ruth Roberts Summer Student Academy fellowship (to A.L.). \n\nAuthor contributions: L.J. and H.T.K. designed research; L.J., T.F., H.P.S., R.Z., A.L., J.H., and H.T.K. performed research; H.P.S., A.M., M.S., and D.A.T. contributed new reagents/analytic tools; L.J., T.F., and H.P.S. analyzed data; and L.J., A.D.R., and H.T.K. wrote the paper.", revision_no = "20", abstract = "The study of hematopoietic colony-forming units using semisolid culture media has greatly advanced the knowledge of hematopoiesis. Here we report that similar methods can be used to study pancreatic colony-forming units. We have developed two pancreatic colony assays that enable quantitative and functional analyses of progenitor-like cells isolated from dissociated adult (2–4 mo old) murine pancreas. We find that a methylcellulose-based semisolid medium containing Matrigel allows growth of duct-like “Ring/Dense” colonies from a rare (∼1%) population of total pancreatic single cells. With the addition of roof plate-specific spondin 1, a wingless-int agonist, Ring/Dense colony-forming cells can be expanded more than 100,000-fold when serially dissociated and replated in the presence of Matrigel. When cells grown in Matrigel are then transferred to a Matrigel-free semisolid medium with a unique laminin-based hydrogel, some cells grow and differentiate into another type of colony, which we name “Endocrine/Acinar.” These Endocrine/Acinar colonies are comprised mostly of endocrine- and acinar-like cells, as ascertained by RNA expression analysis, immunohistochemistry, and electron microscopy. Most Endocrine/Acinar colonies contain beta-like cells that secrete insulin/C-peptide in response to D-glucose and theophylline. These results demonstrate robust self-renewal and differentiation of adult Ring/Dense colony-forming units in vitro and suggest an approach to producing beta-like cells for cell replacement of type 1 diabetes. The methods described, which include microfluidic expression analysis of single cells and colonies, should also advance study of pancreas development and pancreatic progenitor cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37763, title ="A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins", author = "Mahdavi, Alborz and Segall-Shapiro, Thomas H.", journal = "Journal of the American Chemical Society", volume = "135", number = "8", pages = "2979-2982", month = "February", year = "2013", doi = "10.1021/ja400448f", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130404-103625840", note = "© 2013 American Chemical Society. \n\nReceived: January 15, 2013; Published: February 13, 2013. \n\nWe are grateful for financial support by National Institutes of Health grant NIH RO1 GM062523 and the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation (D.A.T.), the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from U.S. Army Research Office (D.A.T. and R.F.I.), the American Heart Association (J.J.S.), and the Robert A. Welch Foundation (J.J.S.). A.M. was supported by a scholarship from the National Science and Engineering Research Council of Canada and by a postgraduate scholarship from the Donna and Benjamin M. Rosen Center for Bioengineering at Caltech.", revision_no = "23", abstract = "We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide–alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/38546, title ="Peptide and Protein Hydrogels", author = "Dooling, Lawrence J. and Tirrell, David A.", number = "11", pages = "93-124", month = "January", year = "2013", doi = "10.1039/9781849735629-00093", isbn = "9781849735612", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130516-133709530", note = "© 2013 The Royal Society of Chemistry. \n\nWork on protein hydrogels at Caltech is supported by NIH grant U01 DK089533-01.", revision_no = "17", abstract = "Proteins are a fascinating class of macromolecules from both functional and structural perspectives. They catalyze the reactions that sustain life, bind ligands with high affinity and specificity, and mediate interactions among biomolecules in complex cellular milieux. Proteins also assemble into higher-order structures that arc responsible for the mechanical integrity of cells and tissues. Their diverse functional and structural properties have made proteins important building blocks in the development of new biomaterials.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/48818, title ="The Wonder of Life in Its Chemical Aspect", author = "Tirrell, David A.", number = "261", pages = "199-210", month = "January", year = "2013", doi = "10.1007/12_2013_261", issn = "0065-3195", isbn = "978-3-319-01136-3", url = "https://resolver.caltech.edu/CaltechAUTHORS:20140822-131055629", note = "© 2013 Springer-Verlag Berlin Heidelberg.\n\nPublished online: 27 November 2013.", revision_no = "16", abstract = "Hermann Staudinger was deeply interested in both macromolecular chemistry and biology. This chapter reviews briefly the shared origins of studies of natural and synthetic polymers, the subsequent divergence of the two fields, and their more recent convergence, made possible by the development of recombinant DNA methodology. The use of recombinant DNA technology to prepare well-defined macromolecular materials is discussed, along with the use of non-canonical amino acids as probes of protein synthesis in complex cellular systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/34214, title ="A magnetic cell-based sensor", author = "Wang, Hua and Mahdavi, Alborz", journal = "Lab on a Chip", volume = "12", number = "21", pages = "4465-4471", month = "August", year = "2012", doi = "10.1039/C2LC40392G ", issn = "1473-0197", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120919-115826812", note = "© 2012 Royal Society of Chemistry.\n\nReceived 23 Apr 2012, Accepted 13 Aug 2012.\nFirst published on the web 14 Aug 2012.\nH. W. was supported by a California Institute of Technology\nInnovation Initiative (CI2) Research Grant. A.M. was supported by a National Science and Engineering Research Council of Canada (NSERC) Scholarship, a post-graduate scholarship by Caltech Donna and Benjamin M. Rosen Center for Bioengineering and the NSF Center for the Science and\nEngineering of Materials at Caltech (NSF DMR 0520565). The\nauthors would like to acknowledge Dr Shouhei Kousai for his\nsupport on the CMOS magnetic sensor chip development; Constantine Sideris for his help on developing the FPGA\nVerilog programs; Alex Pai for his support on building the\nsensor modules; and United Microelectronics Corporation\n(UMC) for providing CMOS sensor chip foundry service.", revision_no = "27", abstract = "Cell-based sensing represents a new paradigm for performing direct and accurate detection of cell- or tissue-specific responses by incorporating living cells or tissues as an integral part of a sensor. Here we report a new magnetic cell-based sensing platform by combining magnetic sensors implemented in the complementary metal-oxide-semiconductor (CMOS) integrated microelectronics process with cardiac progenitor cells that are differentiated directly on-chip. We show that the pulsatile movements of on-chip cardiac progenitor cells can be monitored in a real-time manner. Our work provides a new low-cost approach to enable high-throughput screening systems as used in drug development and hand-held devices for point-of-care (PoC) biomedical diagnostic applications.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/33529, title ="A Quantitative Proteomics Approach to the Identification of Protein Targets of Small Bacterial RNAs", author = "Bagert, John and Tirrell, David", journal = "Protein Science", volume = "21", number = "S1", pages = "76", month = "August", year = "2012", issn = "0961-8368", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120824-153822523", note = "Support for this work was provided by the National\nInstitutes of Health.", revision_no = "11", abstract = "Residue-specific incorporation of non-canonical\namino acids (ncAAs) into cellular proteins allows\ndiscrimination of new proteins from old; labeled proteins\ncan be selectively conjugated to purification tags,\nenriched by affinity chromatography, and identified by\nmass spectrometry. This method, termed bio-orthogonal\nnon-canonical amino acid tagging (BONCAT), is an\nattractive approach for the selective identification of\nproteins produced by a cell in response to biological\ncues. In this work we combined the BONCAT approach\nwith stable isotope labeling by amino acids in cell\nculture (SILAC), to develop a proteomic technique\ncapable not only of enriching but also of quantifying\nthe subset of newly synthesized proteins produced\nduring an ncAA pulse. Armed with this method, we\nconducted a proteomic search for direct regulatory\ntargets of the E. coli small RNA CyaR, a Crp-activated\nregulatory RNA involved in cell metabolism and\nquorum sensing. Following expression of either CyaR\nor a negative RNA control, we pulse-labeled cultures\nwith azidohomoalanine (Aha), a methionine surrogate,\nfor 15 minutes. Aha-labeled proteins were conjugated\nto an alkynyl-biotin affinity tag by copper-catalyzed\nazide-alkyne cycloaddition, purified on streptavidin\nresin, and analyzed by LC/MS-MS. By comparing the\nrelative abundances of proteins produced in the CyaR\nand control cultures, we identified three known CyaR\nprotein targets and a set of 26 candidate proteins as\nsignificantly up or down-regulated. A regulatory RNA\nassay with target-GFP fusions verified a subset of the\ncandidate proteins as directly regulated by CyaR.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/33587, title ="Cell type-specific proteomic profiling in Caenorhabditis elegans", author = "Yuet, Kai and Doma, Meenakshi", journal = "Protein Science", volume = "21", number = "S1", pages = "126", month = "August", year = "2012", issn = "0961-8368", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120828-072510427", revision_no = "15", abstract = "Caenorhabditis elegans is a small, free-living soil\nnematode. For several decades. this multi-cellular\norganism has been used to study intricate biological\nand molecular processes in fields ranging from\ndevelopmental biology to neurobiology. In a complex\neukaryote like C. elegans, cell and tissue heterogeneity\nrestricts the usefulness oflarge-scale, mass spectrometrybased\nanalysis of an organism's proteome. As worm cell\nlines are not available and enriching for specific cells or\ntissues is challenging, researchers cannot systematically\nidentify low abundance proteins expressed in specifc\ncells or tissues from whole-worm lysates. To isolate\nprotein from specific cells, we have engineered a family of mutant C. elegans phenylalanyl-tRNA synthetases\ncapable of appending azide- or alkynyl-bearing analogs\nof phenylalanine to tRNAPhe. As these analogs are\nnot substrates for any of the wild-type aminoacyltRNA\nsynthetases in C. elegans, we achieved cell type\nselectivity by spatially restricting the expression of a\nmutant synthetase using cell type-specific promoters.\nBecause proteins from those cells that express the\nmutant synthetase - and only those cells - contain the\nanalogs, they can be enriched by treatment with alkyneor\nazide-functionalized biotin reagents and subsequent\naffinity chromatography for identification by LC/MSMS.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/34705, title ="Dopaminergic modulation of the hippocampal neuropil proteome identified by bioorthogonal noncanonical amino acid tagging (BONCAT)", author = "Hodas, Jennifer J. L. and Nehring, Anne", journal = "Proteomics", volume = "12", number = "15-16", pages = "2464-2476", month = "August", year = "2012", doi = "10.1002/pmic.201200112 ", issn = "1615-9853", url = "https://resolver.caltech.edu/CaltechAUTHORS:20121005-102359212", note = "© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Received: March 11, 2012.\nRevised: May 21, 2012.\nAccepted: May 22, 2012.\nArticle first published online: 20 Aug. 2012.\nThis work was supported by the German Academy for Natural\nScientists Leopoldina and the DFG (Emmy Noether Program\nand a DIP grant to D. C. D.), the NIH (E. M. S. and D. A. T.\n5R01GM62523–9), HHMI (E. M. S.), Gordon and Betty Moore\nFoundation (M. J. S., S. H.), and the Beckman Institute (M. J.\nS, S. H.).", revision_no = "21", abstract = "Local protein synthesis and its activity-dependent modulation via dopamine receptor stimulation play an important role in synaptic plasticity – allowing synapses to respond dynamically to changes in their activity patterns. We describe here the metabolic labeling, enrichment, and MS-based identification of candidate proteins specifically translated in intact hippocampal neuropil sections upon treatment with the selective D1/D5 receptor agonist SKF81297. Using the noncanonical amino acid azidohomoalanine and click chemistry, we identified over 300 newly synthesized proteins specific to dendrites and axons. Candidates specific for the SKF81297-treated samples were predominantly involved in protein synthesis and synapse-specific functions. Furthermore, we demonstrate a dendrite-specific increase in proteins synthesis upon application of SKF81297. This study provides the first snapshot in the dynamics of the dopaminergic hippocampal neuropil proteome.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/33576, title ="Identifying Protein Targets of a Tumor Suppressor MicroRNA in Human Metastatic Breast Cancer Cells", author = "Lu, Ying Y. and Tirrell, David", journal = "Protein Science", volume = "21", number = "S1", pages = "175", month = "August", year = "2012", issn = "0961-8368", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120827-145247385", revision_no = "12", abstract = "MicroRNAs (miRs) are small non-coding RNAs\nthat are predicted to regulate more than 50% of all\nhuman protein-coding genes by targeting mRNAs\nfor degradation or translational repression. Most\ntargets of miRs are identified by assessing changes\nin the transcriptome. While transcriptome analysis is\nsuccessful in capturing mRNAs that are targeted for\ndegradation, it fails to identify targets regulated via\ntranslational repression in which mRNA abundance is\nunaltered. Therefore, a method to systematically identify\ntargets at the proteomic level is needed. Bio-orthogonal\nnon-canonical amino acid tagging (BONCAT) is a\nmethod that labels the cellular proteome using the cell’s endogenous machinery, co-translationally incorporating\nnon-canonical amino acids into newly synthesized\nproteins. Azidohomoalanine, a methionine analogue,\nhas been used to tag newly synthesized proteins,\npresenting azide groups at methionine positions. An\nalkyne-functionalized purification tag can be attached\nvia copper-catalyzed azide-alkyne cycloaddition\nto selectively enrich newly synthesized proteins\nfrom the pre-existing protein pool, reducing sample\ncomplexity and making the proteome amenable to\ntemporal characterization by mass spectrometry. Here\nwe combine BONCAT with stable isotope labeling\nby amino acids in cell culture (SILAC), to identify\nprotein targets of miR-126 in the human metastatic\nbreast cancer cell line mda-mb-231. MiR-126 has been\nproposed to suppress metastasis, and is down-regulated\nin many types of cancers; however, only a few of its\ntargets have been identified. The results of this study\nwill help elucidate the mechanism of suppression and\nprovide insight into the design of epigenetic therapies\nthat restore expression of tumor suppressor genes.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/33579, title ="Identifying Secreted Proteins by Non-canonical Amino Acid Tagging", author = "Mahdavi, Alborz and Mazmanian, Sarkis", journal = "Protein Science", volume = "21", number = "S1", pages = "151", month = "August", year = "2012", issn = "0961-8368", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120827-160146933", note = "© 2012 The Protein Society.", revision_no = "15", abstract = "Identification of proteins secreted by specific subpopulations\nof cells is an important step in studies of\ncell-cell interaction. Here we describe a method to tag,\nenrich and identify secreted proteins in a cell-selective\nmanner. Labeling is restricted to the cells of interest by\nintroducing a mutant methionyl-tRNA synthetase that\ncharges tRNAMet with azidonorleucine, which can be\ntagged by click chemistry. Proteins made by cells that\ndo not express the mutant synthetase are not labeled.\nApplications of this approach to the identification of\nsecreted proteins will be discussed.", } @conference_item {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/33494, title ="Selenoamino acids and the development of biomaterials responsive to reactive oxygen species (ROS)", author = "Ball, Nicholas D. and Tirrell, David A.", pages = "AEI-64", month = "August", year = "2012", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120823-150207237", revision_no = "12", abstract = "Oxidative stress is a common biol. effect resulting from the imbalance of reactive oxygen species (ROS) generated and depleted in living organisms. This disruption in oxidative species can result in widespread cellular damage with systematic physiol. consequences often assocd. with tissue injury and disease. Herein, we describe methodol. that takes advantage of the unique reactivity of selenium-based amino acids to undergo oxidative elimination to form alkenes and subsequent conjugate addn. with thiols. We exploit this reactivity to develop elastin-based biomaterials that are responsive to reactive oxygen species. We demonstrate this transformation in cross-linked gels and hydrogels, and discuss its application in ROS dosimetry and drug delivery. This research outlines a strategy by our lab. to utilize artificial amino acids to introduce novel reactivity and structural properties in proteins.The oxidn. of selenium compds. to promote unique reactivity in biomaterials serves as a platform for my future research interests in introducing small, oxidized mols. (i.e. SO2 and CO2) into org. mols. This ongoing work compliments my previous research experience in metal catalysis and mechanism. An aspect of my future research will focus on the development of green strategies using metal catalysis to convert biomaterials such as lignin and cellulose to common energy and industrial feedstocks.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/35490, title ="State-Selective Metabolic Labeling of Cellular Proteins", author = "Ngo, John T. and Babin, Brett M.", journal = "ACS Chemical Biology", volume = "7", number = "8", pages = "1326-1330", month = "August", year = "2012", doi = "10.1021/cb300238w ", issn = "1554-8929", url = "https://resolver.caltech.edu/CaltechAUTHORS:20121115-132451309", note = "© 2012 American Chemical Society. Received: May 14, 2012.\nAccepted: June 7, 2012. Published: June 12, 2012.\nThis work was supported by National Institutes of Health grant NIH R01 GM062523 and by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office.", revision_no = "21", abstract = "Transcriptional activity from a specified promoter can provide a useful marker for the physiological state of a cell. Here we introduce a method for selective tagging of proteins made in cells in which specified promoters are active. Tagged proteins can be modified with affinity reagents for enrichment or with fluorescent dyes for visualization. The method allows state-selective analysis of the proteome, whereby proteins synthesized in predetermined physiological states can be identified. The approach is demonstrated by proteome-wide labeling of bacterial proteins upon activation of the P_(BAD) promoter and the SoxRS regulon and provides a basis for analysis of more complex systems including spatially heterogeneous microbial cultures and biofilms.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/34465, title ="Synthesis and Cell Adhesive Properties of Linear and Cyclic RGD Functionalized Polynorbornene Thin Films", author = "Patel, Paresma R. and Kiser, Rosemary Conrad", journal = "Biomacromolecules", volume = "13", number = "8", pages = "2546-2553", month = "August", year = "2012", doi = "10.1021/bm300795y", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120926-114920502", note = "© 2012 American Chemical Society. \n\nReceived: May 21, 2012; Revised: July 5, 2012; Published: July 11, 2012. \n\nWe thank Professor Chin-Lin Guo for use of his inverted epifluorescence microscope, and Mr. Jiun-Yann Yu for assistance with the microscope. Ms. Chithra Krishnamurthy is thanked for helpful discussions. This research was supported by the National Institutes of Health (5R01-GM31332, F32 HL091440), the NSF Materials Research Science and Engineering Center at Caltech (DMR 0520565), the Beckman Institute at Caltech (postdoctoral fellowship to R.C.K.), and grants from the California Institute for Regenerative Medicine. Materia, Inc. is thanked for its donation of metathesis catalysts.", revision_no = "26", abstract = "Described herein is the efficient synthesis and evaluation of bioactive arginine-glycine-aspartic acid (RGD) functionalized polynorbornene-based materials for cell adhesion and spreading. Polynorbornenes containing either linear or cyclic RGD peptides were synthesized by ring-opening metathesis polymerization (ROMP) using the well-defined ruthenium initiator [(H_(2)IMes)(pyr)_(2)(Cl)_(2)Ru═CHPh]. The random copolymerization of three separate norbornene monomers allowed for the incorporation of water-soluble polyethylene glycol (PEG) moieties, RGD cell recognition motifs, and primary amines for postpolymerization cross-linking. Following polymer synthesis, thin-film hydrogels were formed by cross-linking with bis(sulfosuccinimidyl) suberate (BS^3), and the ability of these materials to support human umbilical vein endothelial cell (HUVEC) adhesion and spreading was evaluated and quantified. When compared to control polymers containing either no peptide or a scrambled RDG peptide, polymers with linear or cyclic RGD at varying concentrations displayed excellent cell adhesive properties in both serum-supplemented and serum-free media. Polymers with cyclic RGD side chains maintained cell adhesion and exhibited comparable integrin binding at a 100-fold lower concentration than those carrying linear RGD peptides. The precise control of monomer incorporation enabled by ROMP allows for quantification of the impact of RGD structure and concentration on cell adhesion and spreading. The results presented here will serve to guide future efforts for the design of RGD functionalized materials with applications in surgery, tissue engineering, and regenerative medicine.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/32262, title ="SPSJ International Award for E. W. Meijer, André Persoons, and David A. Tirrell", author = "Meijer, E. W. and Tirrell, D. A.", journal = "Angewandte Chemie International Edition", volume = "51", number = "23", pages = "5535-5535", month = "June", year = "2012", doi = "10.1002/anie.201202984", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120705-092913624", note = "© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.\n\nIssue published online: 30 May 2012; Article first published online: 8 May 2012.\n\n", revision_no = "17", abstract = "The Society for Polymer Science, Japan (SPSJ) International Award was established in 1994, and is the highest honor that the society gives to international scientists over the age of 55 for their contributions to the field and international activities related to the SPSJ. The 2011 International Award winners are Bert Meijer, David A. Tirrell, and André Persoons, who has reported in Angewandte\nChemie on strongly coupled oscillators.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/31999, title ="Two-Strain, Cell-Selective Protein Labeling in Mixed Bacterial Cultures", author = "Truong, Frank and Yoo, Tae Hyeon", journal = "Journal of the American Chemical Society", volume = "134", number = "20", pages = "8551-8556", month = "May", year = "2012", doi = "10.1021/ja3004667", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120621-094016975", note = "© 2012 American Chemical Society. \n\nReceived: January 25, 2012; Published online: May 10, 2012; Published in issue: May 23, 2012. \n\nWe thank John Ngo, Janek Szychowski, Caglar Tanrikulu, and James Van Deventer for helpful discussions, and Felicia Rusnak, Jie Zhou, and Mona Shahgholi for help with spectroscopic measurements. This work was supported by National Institutes of Health Grant NIH R01 GM062523 and by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office.", revision_no = "26", abstract = "Cell-selective metabolic labeling of proteins\nwith noncanonical amino acids enables the study of proteomic\nchanges in specified subpopulations of complex multicellular\nsystems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains cocultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper(I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/29277, title ="Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish", author = "Hinz, Flora I. and Dieterich, Daniela C.", journal = "ACS Chemical Neuroscience", volume = "3", number = "1", pages = "40-49", month = "January", year = "2012", doi = "10.1021/cn2000876 ", issn = "1948-7193", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120214-110853304", note = "© 2011 American Chemical Society. \n\nReceived: September 21, 2011; Accepted: November 7, 2011; Published: November 7, 2011. \n\nThis work was supported by the Max-Planck Society. D.A.T. acknowledges support from NIH (GM62523). F.I.H. acknowledges\nsupport from NIH/NRSA Institutional training grant 5T32 GM07616. \n\nThe authors would like to thank Mark Aizenberg for his help with behavioral tests, Georgi Tushev for his help with calculations of zebrafish proteome methionine content, Jennifer Hodas and John Ngo for general discussions and Stefanie Bunse and Susanne tom Dieck for comments on the manuscript.", revision_no = "21", abstract = "Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce noncanonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells’ own translational machinery. Using the selective “click reaction”, this allows for the identification and visualization of newly synthesized proteins in vitro. Here we demonstrate that noncanonical amino acid labeling can be achieved in vivo in an intact organism capable of simple learning behavior, the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, stimulation with a GABA antagonist that elicits seizures in the larval zebrafish causes an increase in protein synthesis throughout the proteome, which can also be visualized in intact larvae.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53729, title ="Artificial Proteins", author = "Buck, M. E. and Tirrell, D. A.", number = "9", pages = "117-136", month = "January", year = "2012", doi = "10.1016/B978-0-444-53349-4.00219-3", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-140835499", note = "Copyright © 2012 Elsevier. \n\nAvailable online 30 May 2012.", revision_no = "9", abstract = "Recombinant DNA methods provide a powerful, general route to macromolecular systems of controlled chain length, sequence, and stereochemistry. This chapter summarizes recent advances in the synthesis of artificial proteins, including analogs of elastin, silk, and collagen, as well as de novo-designed proteins with no natural counterparts. The principles of protein synthesis are presented briefly, as are the most important methods of genetic engineering and methods for the incorporation of noncanonical amino acids into proteins expressed in microbial cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/29267, title ="EPR Study of Spin Labeled Brush Polymers in Organic Solvents", author = "Xia, Yan and Li, Yongjun", journal = "Journal of the American Chemical Society", volume = "133", number = "49", pages = "19953-19959", month = "December", year = "2011", doi = "10.1021/ja2085349", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20120214-082513251", note = "© 2011 American Chemical Society. \n\nPublication Date (Web): October 24, 2011. Received: September 9, 2011.\n\n We gratefully thank Dr. Xuegong Lei for help with initial EPR experiments. This work was supported by the Department of Energy (DE-FG02-05ER46218) and by the NSF Center for the Science and Engineering of Materials at the California Institute of Technology.", revision_no = "29", abstract = "Spin-labeled polylactide brush polymers were synthesized via ring-opening metathesis polymerization (ROMP), and nitroxide radicals were incorporated at three different locations of brush polymers: the end and the middle of the backbone, and the end of the side chains (periphery). Electron paramagnetic resonance (EPR) was used to quantitatively probe the macromolecular structure of brush polymers in dilute solutions. The peripheral spin-labels showed significantly higher mobility than the backbone labels, and in dimethylsulfoxide (DMSO), the backbone end labels were shown to be more mobile than the middle labels. Reduction of the nitroxide labels by a polymeric reductant revealed location-dependent reactivity of the nitroxide labels: peripheral nitroxides were much more reactive than the backbone nitroxides. In contrast, almost no difference was observed when a small molecule reductant was used. These results reveal that the dense side chains of brush polymers significantly reduce the interaction of the backbone region with external macromolecules, but allow free diffusion of small molecules.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/27786, title ="Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis", author = "Ngo, John T. and Tirrell, David A.", journal = "Accounts of Chemical Research", volume = "44", number = "9", pages = "677-685", month = "September", year = "2011", doi = "10.1021/ar200144y", issn = "0001-4842", url = "https://resolver.caltech.edu/CaltechAUTHORS:20111115-121540025", note = "© 2011 American Chemical Society. \n\nPublished In Issue September 20, 2011; Article ASAP August 04, 2011; Received: May 24, 2011. \n\nWork on noncanonical amino acids at Caltech is supported by the National Institutes of Health (Grant GM 62523) and by the Army Research Office through the Institute for Collaborative Biotechnologies.", revision_no = "27", abstract = "Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein’s gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell.\n\nBecause this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a “pulse” in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins.\n\nNoncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs.\n\nControlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins.\n\nIn this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/27354, title ="A BODIPY-Cyclooctyne for Protein Imaging in Live Cells", author = "Beatty, Kimberly E. and Szychowski, Janek", journal = "ChemBioChem", volume = "12", number = "14", pages = "2137-2139", month = "September", year = "2011", doi = "10.1002/cbic.201100277", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20111021-134710184", note = "© 2011 Wiley-VCH Verlag. Received: May 1, 2011. Published online on August 9, 2011. We thank Julie Liu, Matthew J. Hangauer, Jeremy M. Baskin, Michael S. Boyce, Andrew Mehle, and Carolyn R. Bertozzi for valuable discussions. We are grateful to Mandy K. Vink for preparing Aha and to Stacy A. Maskarinec for providing Rat-1 fibroblasts. Marissa L. Mock, Ying Ying Lu, and Rebecca E. Connor made helpful comments on the manuscript. Imaging was done at the\nBiological Imaging Center (Beckman Institute at Caltech). Flow cytometry was done at Caltech under the guidance of Diana Perez and Rochelle Diamond. Support was provided by a Fannie and John Hertz Foundation fellowship to K.E.B. , by an NIH postdoctoral fellowship (F32M071214) to J.D.F., and by grants from the NIH (R01M062523) and the Army Research Office (W911NF0810227).", revision_no = "22", abstract = "Cellular proteins that bear reactive azides can be imaged by fluorescence microscopy following strain-promoted ligation to cyclooctyne dyes. Here we describe BODIPY-cyclooctyne (BDPY), a membrane-permeant fluorophore that can be used to label intracellular proteins in live mammalian cells. Flow cytometry reveals fluorescence signals more than 25-fold above background after labeling of azide-tagged cells with BDPY.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/37528, title ="Three-dimensional traction force microscopy for studying\ncellular interactions with biomaterials", author = "Notbohm, J. and Kim, J.-H.", journal = "Procedia IUTAM", volume = "4", pages = "144-150", month = "July", year = "2011", issn = "2210-9838", url = "https://resolver.caltech.edu/CaltechAUTHORS:20130315-092735718", note = "© 2012 Published by Elsevier Ltd. This work was funded by a grant from the National Science Foundation through the Center for the Science and Engineering of Materials at the California Institute of Technology. J.N. was supported by the\nDepartment of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship\n(NDSEG) Program.", revision_no = "20", abstract = "The interactions between biochemical and mechanical signals during cell adhesion, migration, spreading and other processes influence cellular behavior. Three-dimensional measurement techniques are needed to investigate the effect of mechanical properties of the substrate on cellular behavior. This paper discusses a three-dimensional full-field measurement technique that has been developed for measuring large deformations in soft materials. The technique utilizes a digital volume correlation (DVC) algorithm to track motions of sub-volumes within 3-D images obtained using laser scanning confocal microscopy. The technique is well-suited for investigating 3-D mechanical interactions between cells and the extracellular matrix and for obtaining local constitutive properties of soft biomaterials. Results from the migration of single fibroblast cells on polyacrylamide gels and their implications for cell motility models are discussed. The implications that the traction distributions of epithelial cell clusters have on the inhibition of proliferation due to cell contact and scattering of cells in a cluster are discussed. These results provide insights on force fields generated by cells and the role of the mechanical properties of the substrate on cellular interactions and mechanotransduction. Analytical solutions and finite element simulations are used to elucidate the mechanics of cellular forces exerted on the extracellular matrix.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/23496, title ="Three-Dimensional Traction Force Microscopy: A New Tool for Quantifying Cell-Matrix Interactions", author = "Franck, Christian and Maskarinec, Stacey A.", journal = "PLoS ONE", volume = "6", number = "3", pages = "Art. No. e17833", month = "March", year = "2011", doi = "10.1371/journal.pone.0017833 ", issn = "1932-6203", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110429-100954190", note = "© 2011 Franck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. \n\nReceived September 24, 2010; Accepted February 15, 2011; Published March 29, 2011. \n\nEditor: Igor Sokolov, Clarkson University, United States of America. \n\nThis work was supported by the National Science Foundation (DMR # 0520565) through the Center for Science and Engineering of Materials (CSEM) at the California Institute of Technology. S.A.M. had additional support from a predoctoral National Research Service Award fellowship from the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. \n\nThe authors thank Professor S.E. Fraser’s laboratory for providing GFP-transfected 3T3 fibroblasts and Petros Arakelin for his technical assistance. \n\nAuthor Contributions:\nConceived and designed the experiments: CF SAM DAT GR. Performed\nthe experiments: CF SAM. Analyzed the data: CF SAM DAT GR.\nContributed reagents/materials/analysis tools: CF SAM. Wrote the paper:\nCF SAM DAT GR.", revision_no = "20", abstract = "The interactions between biochemical processes and mechanical signaling play important roles during various cellular processes such as wound healing, embryogenesis, metastasis, and cell migration. While traditional traction force measurements have provided quantitative information about cell matrix interactions in two dimensions, recent studies have shown significant differences in the behavior and morphology of cells when placed in three-dimensional environments. Hence new quantitative experimental techniques are needed to accurately determine cell traction forces in three dimensions. Recently, two approaches both based on laser scanning confocal microscopy have emerged to address this need. This study highlights the details, implementation and advantages of such a three-dimensional imaging methodology with the capability to compute cellular traction forces dynamically during cell migration and locomotion. An application of this newly developed three-dimensional traction force microscopy (3D TFM) technique to single cell migration studies of 3T3 fibroblasts is presented to show that this methodology offers a new quantitative vantage point to investigate the three-dimensional nature of cell-ECM interactions.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/23237, title ="Homoisoleucine: A Translationally Active Leucine Surrogate of Expanded Hydrophobic Surface Area", author = "Van Deventer, James A. and Fisk, John D.", journal = "ChemBioChem", volume = "12", number = "5", pages = "700-702", month = "March", year = "2011", doi = "10.1002/cbic.201000731", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110404-112939537", note = "© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. \n\nReceived: December 9, 2010; Published online on February 15, 2011. \n\nWe thank Shelly Tzlil for assistance in implementing the model for thermal unfolding of coiled-coil peptides in Matlab. This research was supported by NIH grant GM62523. J.A.V. was supported in part by an N.D.S.E.G. Fellowship; J.D.F. was supported in part by an NIH Postdoctoral Fellowship.", revision_no = "24", abstract = "Hil of a strong peptide! Homoisoleucine (Hil) serves as an effective surrogate for leucine with respect to protein translation in bacterial cells. Replacement of Leu by Hil stabilizes coiled-coil peptides, as shown by the elevation of the thermal denaturation temperature. The increase in denaturation temperature is larger than that observed previously for replacement of Leu by trifluoroleucine.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/21884, title ="2010 Panel on the Biomaterials Grand Challenges", author = "Reichert, William “Monty” and Ratner, Buddy D.", journal = "Journal of Biomedical Materials Research. Part A", volume = "96A", number = "2", pages = "275-287", month = "February", year = "2011", doi = "10.1002/jbm.a.32969 ", issn = "1549-3296", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110125-134801443", note = "© 2010 Wiley Periodicals, Inc.\nReceived 3 August 2010; accepted 24 August 2010.\nPublished online 29 November 2010 in Wiley Online Library.\n\nWilliam ‘‘Monty’’ Reichert is the Panel organizer and Buddy D. Ratner, James Anderson, Art Coury, Allan S. Hoffman, Cato T. Laurencin, and\nDavid Tirrell are the Panelists of 2010 Panel on the Biomaterials Grand Challenges.", revision_no = "16", abstract = "In 2009, the National Academy for Engineering issued the Grand Challenges for Engineering in the 21st Century comprised of 14 technical challenges that must be addressed to build a healthy, profitable, sustainable, and secure global community (http://www.engineeringchallenges.org). Although crucial, none of the NEA Grand Challenges adequately addressed the challenges that face the biomaterials community. In response to the NAE Grand Challenges, Monty Reichert of Duke University organized a panel entitled Grand Challenges in Biomaterials at the at the 2010 Society for Biomaterials Annual Meeting in Seattle. Six members of the National Academies—Buddy Ratner, James Anderson, Allan Hoffman, Art Coury, Cato Laurencin, and David Tirrell—were asked to propose a grand challenge to the audience that, if met, would significantly impact the future of biomaterials and medical devices. Successfully meeting these challenges will speed the 60-plus year transition from commodity, off-the-shelf biomaterials to bioengineered chemistries, and biomaterial devices that will significantly advance our ability to address patient needs and also to create new market opportunities.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/23013, title ="Core-Clickable PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP: Grafting-Through and Clicking-To", author = "Johnson, Jeremiah A. and Lu, Ying Y.", journal = "Journal of the American Chemical Society", volume = "133", number = "3", pages = "559-566", month = "January", year = "2011", doi = "10.1021/ja108441d ", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110321-113913494", note = "© 2010 American Chemical Society. \n\nReceived: September 18, 2010. Article ASAP December 13, 2010. Published In Issue January 26, 2011. \n\nWe thank Dr. S. Virgil and Mr. S. Presolski for helpful advice. This work was supported by the National Institutes of Health (NIH, R01-GM31332), the Beckman Institute at Caltech (postdoctoral fellowship for J.A.J.), and the MRSEC program of the National Science Foundation (NSF) under Award No. DMR-0520565.", revision_no = "28", abstract = "The combination of highly efficient polymerizations with modular \"click\" coupling reactions has enabled the synthesis of a wide variety of novel nanoscopic tructures. Here we demonstrate the facile synthesis of a new class of clickable, branched nanostructures, polyethylene glycol (PEG)-branch-azide bivalent-brush polymers, facilitated by \"graft-through\" ring-opening metathesis polymerization of a branched norbornene-PEG-chloride macromonomer followed by halide-azide exchange. The resulting bivalent-brush polymers possess azide groups at the core near a polynorbornene backbone with PEG chains extended into solution; the structure resembles a unimolecular micelle. We demonstrate copper-catalyzed azide-alkre cycloaddition (CuAAC) \"click-to\" coupling of a photocleavable doxorubicin (DOX)-alkyne derivative to the azide core. The CuAAC coupling was quantitative across a wide range of nanoscopic sizes (similar to 6-similar to 50 nrn); UV photolysis of the resulting DOX-loaded materials yielded free DOX that was therapeutically effective against human cancer cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/22639, title ="Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition", author = "Szychowski, Janek and Mahdavi, Alborz", journal = "Journal of the American Chemical Society", volume = "132", number = "51", pages = "18351-18360", month = "December", year = "2010", doi = "10.1021/ja1083909 ", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110303-113837195", note = "© 2010 American Chemical Society. \n\nReceived September 16, 2010, Publication Date (Web): December 8, 2010. \n\nThis work was supported by NIH GM62523 (DAT), NIH MH065537 (EMS), the ARO Institute for Collaborative Biotechnologies, and Fonds Québécois de la Recherche sur la Nature et les Technologies (as a postdoctoral fellowship to J.S.). A.M. was supported in part by a National Science and Engineering Council of Canada (NSERC) postgraduate scholarship. We thank Sonja Hess, Jacob Bitterman, Eva Wurster, and Christiane Kowatsch for helpful discussions, as well as the Proteome Exploration Laboratory (PEL), MS, and NMR facilities of the California Institute of Technology. \n\nSequence and mass spectrum of protein 18; ^1H NMR spectra for compounds 4, 5, 6, 9, 11, 12, 14a, and 14b; ^(13)C NMR spectra for compounds 4, 5, 6, 9, 12, 14a, and 14b; MS/MS spectra of tryptic fragments.", revision_no = "32", abstract = "The azide−alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO_2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/23151, title ="Drug-Loaded, Bivalent-Bottle-Brush Polymers by Graft-through ROMP", author = "Johnson, Jeremiah A. and Lu, Ying Y.", journal = "Macromolecules", volume = "43", number = "24", pages = "10326-10335", month = "December", year = "2010", doi = "10.1021/ma1021506 ", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110329-095607608", note = "© 2010 American Chemical Society. \n\nReceived: September 15, 2010; Revised: November 08, 2010. Article ASAP December 02, 2010. Published In Issue December 28, 2010. \n\nWe thank Dr. S. Virgil for helpful discussion and advice. We also thank the Beckman Institute for a postdoctoral fellowship for JAJ. UV-vis experiments were performed in the Beckman Institute Laser Center. This work was supported by the National Institutes of Health (NIH, R01-GM31332), the MRSEC program of the National Science Foundation (NSF) under award number DMR-0520565, and the NSF Center for Chemical Innovation (Powering the Planet, CHE-0802907 and CHE-0947829).", revision_no = "28", abstract = "Graft through ring-opening metathesis polymerization (ROMP) using ruthenium N heterocyclic carbene catalysts has enabled the synthesis of bottle-brush polymers with unprecedented ease and control Here we report the first bivalent-brush polymers, these materials were prepared by graft through ROMP of drug-loaded poly(ethylene glycol) (PEG) based macromonomers (MMs) Anticancer drugs doxorubicin (DOX) and camptothecin (CT) were attached to a norbornene alkyne-PEG MM via a photocleavable linker ROMP of either or both drug loaded MMs generated brush homo and copolymers with low polydispersities and defined molecular weights. Release of free DOX and CT from these materials was initiated by exposure to 365 nm light All of the CT and DOX polymers were at least 10 fold more toxic to human cancer cells after photoinitiated drug release while a copolymer carrying both CT and DOX displayed 30-fold increased toxicity upon irradiation Graft through ROMP of drug loaded macromonomers provides a general method for the systematic study of structure function relationships for stimuli responsive polymers in biological systems.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/21961, title ="Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains", author = "Fong, Eileen and Tirrell, David A.", journal = "Advanced Materials", volume = "22", number = "46", pages = "5271-5275 ", month = "December", year = "2010", doi = "10.1002/adma.201002448 ", issn = "0935-9648", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110202-085127824", note = "© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. \n\nReceived: July 7, 2010; Revised: August 11, 2010; Published online: September 30, 2010. \n\nThe authors thank Drs. Stacey Maskarinec and Shelly Tzlil for discussions. We acknowledge Dr. Anand Asthagiri for Rat-1 fibroblasts and generous access to the fluorescence microscope. E. F. is supported in part by the Nanyang Overseas Scholarship, Singapore. This work is supported by NIH EB001971, by the NSF Center for Science and Engineering of Materials, and by the Armed Forces Institute for Regenerative Medicine Wake Forest–Pittsburgh Consortium.", revision_no = "37", abstract = "Protein-based biomaterials that promote rapid wound healing are prepared by expression of artificial genes in bacterial cells. Artificial extracellular matrix proteins containing full-length fibronectin domains 9 and 10 exhibit strong α_5β_1 integrin binding and support rapid spreading, proliferation, and collective migration of fibroblasts. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/22438, title ="Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications", author = "Johnson, Jeremiah A. and Lu, Ying Y.", journal = "Current Opinion in Chemical Biology", volume = "14", number = "6", pages = "774-780", month = "December", year = "2010", doi = "10.1016/j.cbpa.2010.09.013 ", issn = "1367-5931", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110222-151826904", note = "© 2010 Elsevier Ltd. \n\nAvailable online 9 November 2010. \n\nWork at Caltech on non-canonical amino acids is supported by NIH grant GM62523 and by the Institute for Collaborative Biotechnologies under contract W911NF-09-D-0001 with the Army Research Office.", revision_no = "27", abstract = "Residue-specific incorporation of non-canonical amino acids into proteins allows facile alteration and enhancement of protein properties. In this review, we describe recent technical developments and applications of residue-specific incorporation to problems ranging from elucidation of biochemical mechanisms to engineering of protein-based biomaterials. We hope to inform the reader of the ease and broad utility of residue-specific non-canonical amino acid incorporation with the goal of inspiring investigators outside the field to consider applying this tool to their own research.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/21191, title ="Boundary crossing in epithelial wound healing\n", author = "Fong, Eileen and Tzlil, Shelly", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "107", number = "45", pages = "19302-19307", month = "November", year = "2010", doi = "10.1073/pnas.1008291107 ", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20101206-122849107", note = "© 2010 National Academy of Sciences. \n\nEdited by Steven Boxer, Stanford University, Stanford, CA, and approved September 7, 2010 (received for review June 11, 2010). \n\nWe acknowledge Drs. Julie Liu, Paul Nowatzki, and Stacey Maskarinec for help with protein expression and cell culture. We also\nthank Dr. Zhen-Gang Wang for useful advice on the simulation model. E.F. is supported by the Nanyang Overseas Scholarship, Singapore; S.T. is supported by the Human Frontier Science Program Cross-Disciplinary fellowship. This work is funded by the National Science Foundation Center for the Science and Engineering of Materials at Caltech and by National Institutes of Health Grant EB1971.\n\nAuthor contributions: E.F., S.T., and D.A.T. designed research; E.F. and S.T. performed research; E.F., S.T., and D.A.T. analyzed data; and E.F., S.T., and D.A.T. wrote the paper.", revision_no = "33", abstract = "The processes of wound healing and collective cell migration have been studied for decades. Intensive research has been devoted to understanding the mechanisms involved in wound healing, but the role of cell-substrate interactions is still not thoroughly understood. Here we probe the role of cell-substrate interactions by examining in vitro the healing of monolayers of human corneal epithelial (HCE) cells cultured on artificial extracellular matrix (aECM) proteins. We find that the rate of wound healing is dependent on the concentration of fibronectin-derived (RGD) cell-adhesion ligands in the aECM substrate. The wound closure rate varies nearly sixfold on the substrates examined, despite the fact that the rates of migration and proliferation of individual cells show little sensitivity to the RGD concentration (which varies 40-fold). To explain this apparent contradiction, we study collective migration by means of a dynamic Monte Carlo simulation. The cells in the simulation spread, retract, and proliferate with probabilities obtained from a simple phenomenological model. The results indicate that the overall wound closure rate is determined primarily by the rate at which cells cross the boundary between the aECM protein and the matrix deposited under the cell sheet. \n\n", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/21204, title ="Yielding Behavior in Injectable Hydrogels from Telechelic Proteins", author = "Olsen, Bradley D. and Kornfield, Julia A.", journal = "Macromolecules", volume = "43", number = "21", pages = "9094-9099", month = "November", year = "2010", doi = "10.1021/ma101434a ", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:20101207-081608961", note = "© 2010 American Chemical Society. \n\nReceived June 28, 2010; Revised Manuscript Received September 27, 2010. Published on Web 10/13/2010. \n\nThis work was supported by the NSF Center for the Science and Engineering of Materials and NIH Grant EB1971. B.D.O. was supported by Award F32GM0834 from the National Institute of General Medical Sciences and by a Beckman Institute Postdoctoral Fellowship. We thank Professor S. E. Fraser for suggesting the method of flow visualization reported in Figure 3.", revision_no = "27", abstract = "Injectable hydrogels show substantial promise for use in minimally invasive tissue engineering and drug delivery procedures. A new injectable hydrogel material, developed from recombinant telechelic proteins expressed in E. coli, demonstrates shear thinning by 3 orders of magnitude at large strains. Large-amplitude oscillatory shear illustrates that shear thinning is due to yielding within the bulk of the gel, and the rheological response and flow profiles are consistent with a shear-banding mechanism for yielding. The sharp yielding transition and large magnitude of the apparent shear thinning allow gels to be injected through narrow gauge needles with only gentle hand pressure. After injection the gels reset to full elastic strength in seconds due to rapid re-formation of the physical network junctions, allowing self-supporting structures to be formed. The shear thinning and recovery behavior is largely independent of the midblock length, enabling genetic engineering to be used to control the equilibrium modulus of the gel without loss of the characteristic yielding behavior. The shear-banding mechanism localizes deformation during flow into narrow regions of the gels, allowing more than 95% of seeded cells to survive the injection process.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/21196, title ="Live-Cell Imaging of Cellular Proteins by a Strain-Promoted Azide–Alkyne Cycloaddition\n", author = "Beatty, Kimberly E. and Fisk, John D.", journal = "ChemBioChem", volume = "11", number = "15", pages = "2092-2095", month = "October", year = "2010", doi = "10.1002/cbic.201000419 ", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20101206-145110598", note = "© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. \n\nArticle first published online: 10 Sep. 2010. \n\nWe thank Rebecca E. Connor, Julie C. Liu, Marissa L. Mock, Tae Hyeon Yoo, Jay A. Codelli, and Brian C. Dickinson for advice and assistance. We thank Scott E. Fraser, Chris Waters, and the Biological Imaging Center of the Beckman Institute at Caltech for advice on microscopy, Mona Shahgholi for assistance with mass spectrometry, and Rochelle Diamond and Diana Perez for assistance with flow cytometry. We thank Mandy K. S. Vink for Aha and Stacey A. Maskarinec for cell lines. This work was supported by NIH grant GM62523 to D.A.T. and GM66047 to C.R.B., by the ARO Institute for Collaborative Biotechnologies, by a Hertz Foundation Fellowship and PEO Scholar Award to K.E.B. , by NIH postdoctoral fellowships to J.D.F. and B.P.S., an NSF and NDSEG fellowship to J.M.B., and an NDSEG fellowship to M.J.H.", revision_no = "31", abstract = "Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/20519, title ="Hydration dynamics at fluorinated protein surfaces", author = "Kwon, Oh-Hoon and Yoo, Tae Hyeon", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "107", number = "40", pages = "17101-17106", month = "October", year = "2010", doi = "10.1073/pnas.1011569107 ", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20101026-075632113", note = "© 2010 National Academy of Sciences. \n\nContributed by David A. Tirrell, August 4, 2010 (sent for review May 10, 2010). \n\nWe thank Prof. Thomas Miller for helpful discussion, J.D. Fisk for synthesis of homoisoleucine, and the referees for their thoughtful comments on the original manuscript. This work is supported by National Institutes of Health (NIH) Grant GM62523, National Science Foundation (NSF) Grant DMR-0964886, Office of Naval Research (ONR) Grant N00014-03-1-0793, a Samsung Scholarship (to T.H.Y.), and a National Defense Science and Engineering Graduate Fellowship (to J.A.V.D.). \n\nAuthor contributions: O.-H.K., T.H.Y., C.M.O., J.A.V.D., D.A.T., and A.H.Z. designed research; O.-H.K., T.H.Y., C.M.O., J.A.V.D., D.A.T., and A.H.Z. performed research; O.-H.K., T.H.Y., C.M.O., J.A.V.D., D.A.T., and A.H.Z. analyzed data; and O.-H.K., T.H.Y., C.M.O., J.A.V.D., D.A.T., and A.H.Z. wrote the paper. \n\nO.-H.K. and T.H.Y. contributed equally to this work. \n\nThe authors declare no conflict of interest. \n\nThis article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011569107/-/DCSupplemental.", revision_no = "21", abstract = "Water-protein interactions dictate many processes crucial to protein function including folding, dynamics, interactions with other biomolecules, and enzymatic catalysis. Here we examine the effect of surface fluorination on water-protein interactions. Modification of designed coiled-coil proteins by incorporation of 5,5,5-trifluoroleucine or (4S)-2-amino-4-methylhexanoic acid enables systematic examination of the effects of side-chain volume and fluorination on solvation dynamics. Using ultrafast fluorescence spectroscopy, we find that fluorinated side chains exert electrostatic drag on neighboring water molecules, slowing water motion at the protein surface.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/19032, title ="In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons", author = "Dieterich, Daniela C. and Hodas, Jennifer J. L.", journal = "Nature Neuroscience", volume = "13", number = "7", pages = "897-905", month = "July", year = "2010", doi = "10.1038/nn.2580 ", issn = "1097-6256", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100713-132314376", note = "© 2010 Nature Publishing Group, a division of Macmillan Publishers Limited. \n\nReceived 22 January; accepted 17 May; published online 13 June 2010. \n\nWe thank L. Chen for making beautiful cultured hippocampal neurons. We thank A.J. Link for discussions and help with the tag syntheses. We are grateful to O. Kobler for help with Imaris software. We are extremely grateful to both C. Bertozzi and J. Baskin for providing the difluorinated cyclooctyne-biotin and advising on its use. This work was supported by the German Academy for Natural Scientists Leopoldina (D.C.D.), the US National Institutes of Health (E.M.S. and D.A.T.), the Howard Hughes Medical Institute (E.M.S.), the Ministère de l’Enseignement Supérieur et de la Recherche (G.G.) and the Nationale de la Recherche MorphoSynDiff–INSERM (A.T.). \n\nAuthor Contributions: D.C.D., J.J.L.H., G.G. and E.M.S. performed experiments; D.C.D., G.G., A.T. and E.M.S. designed experiments; D.C.D., J.J.L.H., I.Y.S., G.G. and E.M.S. analyzed data; D.C.D., G.G., A.T. and E.M.S. wrote the paper; J.T.N. and D.A.T. provided reagents.", revision_no = "24", abstract = "Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/17291, title ="Quantifying cellular traction forces in three dimensions", author = "Maskarinec, Stacey A. and Franck, Christian", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "106", number = "52", pages = "22108-22113", month = "December", year = "2009", doi = "10.1073/pnas.0904565106", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100122-134235353", note = "© 2009 by the National Academy of Sciences.\nEdited by Steven G. Boxer, Stanford University, Stanford, CA, and approved November 2, 2009 (received for review April 27, 2009).\nPublished online before print December 15, 2009.\nAuthor contributions: S.A.M., C.F., D.A.T., and G.R. designed research; S.A.M. and C.F.\nperformed research; S.A.M. and C.F. contributed new reagents/analytic tools; S.A.M., C.F.,\nD.A.T., and G.R. analyzed data; and S.A.M., C.F., D.A.T., and G.R. wrote the paper.\nWe thank Petros Arakelian for help in constructing the\nexperimental setup and Professor Scott Fraser’s laboratory for providing the\nGFP-transfected fibroblasts. This work was funded by a grant from the National\nScience Foundation through the Center for the Science and Engineering of\nMaterials at the California Institute of Technology. S.A.M. acknowledges support\nfrom the National Institutes of Health through a predoctoral National Research\nService Award fellowship.", revision_no = "22", abstract = "Cells engage in mechanical force exchange with their extracellular environment through tension generated by the cytoskeleton. A method combining laser scanning confocal microscopy (LSCM) and digital volume correlation (DVC) enables tracking and quantification of cell-mediated deformation of the extracellular matrix in all three spatial dimensions. Time-lapse confocal imaging of migrating 3T3 fibroblasts on fibronectin (FN)-modified polyacrylamide gels of varying thickness reveals significant in-plane (x, y) and normal (z) displacements, and illustrates the extent to which cells, even in nominally two-dimensional (2-D) environments, explore their surroundings in all three dimensions. The magnitudes of the measured displacements are independent of the elastic moduli of the gels. Analysis of the normal displacement profiles suggests that normal forces play important roles even in 2-D cell migration. \n\n", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/17305, title ="Switching from an Induced-Fit to a Lock-and-Key Mechanism in an Aminoacyl-tRNA Synthetase with Modified Specificity\n", author = "Schmitt, Emmanuelle and Tanrikulu, I. Caglar", journal = "Journal of Molecular Biology", volume = "394", number = "5", pages = "843-851", month = "December", year = "2009", doi = "10.1016/j.jmb.2009.10.016 ", issn = "0022-2836", url = "https://resolver.caltech.edu/CaltechAUTHORS:20100126-093702438", note = "© 2009 Elsevier Ltd. \n\nReceived 24 July 2009; revised 8 October 2009; accepted 9 October 2009. Edited by J. Doudna. Available online 16 October 2009. \n\nWe acknowledge the ESRF for providing the X-ray facilities on beamline ID14-2 as well as the synchrotron SOLEIL for providing the X-ray facilities on beamline Proxima I. We thank Centre National de la Recherche Scientifique and Ecole Polytechnique for financial support to the “Unité Mixte de Recherche 7654”. Work at Caltech was supported by National Institutes of Health Grant GM62523 and by the Army Research Office through the Institute for Collaborative Biotechnologies.", revision_no = "21", abstract = "Methionyl-tRNA synthetase (MetRS) specifically binds its methionine substrate in an induced-fit mechanism, with methionine binding causing large rearrangements. Mutated MetRS able to efficiently aminoacylate the methionine (Met) analog azidonorleucine (Anl) have been identified by saturation mutagenesis combined with in vivo screening procedures. Here, the crystal structure of such a mutated MetRS was determined in the apo form as well as complexed with Met or Anl (1.4 to 1.7 Å resolution) to reveal the structural basis for the altered specificity. The mutations result in both the loss of important contacts with Met and the creation of new contacts with Anl, thereby explaining the specificity shift. Surprisingly, the conformation induced by Met binding in wild-type MetRS already occurs in the apo form of the mutant enzyme. Therefore, the mutations cause the enzyme to switch from an induced-fit mechanism to a lock-and-key one, thereby enhancing its catalytic efficiency.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/16886, title ="Generation of Surface-Bound Multicomponent Protein Gradients", author = "Zhang, Kechun and Sugawara, Ayae", journal = "ChemBioChem", volume = "10", number = "16", pages = "2617-2619", month = "November", year = "2009", doi = "10.1002/cbic.200900542 ", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20091207-100800458", note = "© 2009 WILEY. \n\nReceived: 28 August 2009; published Online: 24 Sep 2009. \n\nThis work was supported by the NSF-sponsored Center for the Science and Engineering of Materials at the California Institute of Technology, and by NIH grant GM62523.", revision_no = "28", abstract = "Spatial control of bioactive ligands is achieved by integrating microfluidics and protein engineering. The proteins of interest are mixed in a gradient generator and immobilized on artificial polypeptide scaffolds through the strong association of heterodimeric ZE/ZR leucine zipper pairs. Protein densities and gradient shapes are easily controlled and varied in this method.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/16297, title ="Cell-selective metabolic labeling of proteins", author = "Ngo, John T. and Champion, Julie A.", journal = "Nature Chemical Biology", volume = "5", number = "10", pages = "715-717", month = "October", year = "2009", doi = "10.1038/nchembio.200", issn = "1552-4450", url = "https://resolver.caltech.edu/CaltechAUTHORS:20091013-093448078", note = "© 2009 Nature Publishing Group. \n\nReceived 28 October 2008; Accepted 28 May 2009; Published online 9 August 2009. \n\nWe gratefully acknowledge the US National Institutes of Health (R21 DA020589 and R01 GM62523) and the US Army Research Office Institute for Collaborative Biotechnologies for support of this work. We thank K. Boulware for assistance in generating the three-dimensional movie of infected macrophages. E.M.S. is an investigator of the Howard Hughes Medical Institute. \n\nNote: Supplementary information and chemical compound information is available on the Nature Chemical Biology website.", revision_no = "27", abstract = "Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/16011, title ="Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo", author = "Tanrikulu, I. Caglar and Schmitt, Emmanuelle", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "106", number = "36", pages = "15285-15290", month = "September", year = "2009", doi = "10.1073/pnas.0905735106", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090923-143133627", note = "© 2009 by the National Academy of Sciences. \n\nEdited by Paul R. Schimmel, The Skaggs Institute for Chemical Biology, La Jolla, CA, and approved July 13, 2009 (received for review May 22, 2009). Published online before print August 17, 2009, doi: 10.1073/pnas.0905735106 \n\nWe thank Mona Shahgholi for expert assistance with mass spectrometry, Haick Issaian for assistance with radioisotope use, and Professor Jared Leadbetter for use of his scintillation counter. We thank I. Kwon, A. J. Link, T. H. Yoo, J. D. Fisk, R. E. Connor, M. L. Mock, E. J. Choi and J. T. Ngo for helpful discussions and Dr. J. A. Champion for reviewing the manuscript. This work was supported by National Institutes of Health Grant GM62523 and by the Army Research Office through the Institute for Collaborative Biotechnologies. \n\nAuthor contributions: I.C.T., W.A.G., and D.A.T. designed research; I.C.T. performed research; I.C.T., E.S., Y.M., W.A.G., and D.A.T. analyzed data; and I.C.T. and D.A.T. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/cgi/content/full/0905735106/DCSupplemental.", revision_no = "23", abstract = "Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/16247, title ="Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase", author = "Tang, Yi and Wang, Pin", journal = "ChemBioChem", volume = "10", number = "13", pages = "2188-2190", month = "September", year = "2009", doi = "10.1002/cbic.200900407", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20091012-125839216", note = "Copyright © 2009 WILEY-VCH. \n\nReceived: July 3, 2009. Published online on August 7, 2009. \n\nWe thank Dr. Mona Shahgholi for assistance with MALDI-TOF mass spectrometry. This work was supported by NIH Grant R01-GM62523 and by the NSF Center for the Science and Engineering of Materials at Caltech. Y.T. was supported by a fellowship from the Whitaker Foundation, J.A.V. was supported by a National Defense Science and Engineering Graduate Fellowship, and A.J.L. was supported by a National Science Foundation Graduate Research Fellowship. \n\nDetailed experimental protocols can be found in the Supporting Information.", revision_no = "22", abstract = "Like leucine? A leucine analogue containing a ketone has been incorporated into proteins in E. coli. Only E. coli strains overexpressing an editing-deficient leucyl-tRNA synthetase were capable of synthesizing proteins with the aliphatic ketone amino acid. Modification of ketone-containing proteins under mild conditions has been demonstrated.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/15954, title ="Biosynthesis and Stability of Coiled-Coil Peptides Containing (2S,4R)-5,5,5-Trifluoroleucine and (2S,4S)-5,5,5-Trifluoroleucine", author = "Montclare, Jin Kim and Son, Soojin", journal = "ChemBioChem", volume = "10", number = "1", pages = "84-86", month = "January", year = "2009", doi = "10.1002/cbic.200800164", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090918-093938869", note = "© 2009 Wiley. \n\nThis work was supported by the NIH grants GM62523 and 5FM GM67375-2 (D.A.T. and J.K.M.), and GM65500 (K.K.), NSF graduate fellowship (S.S.), GAANN fellowship (G.A.C.), and a NSF CAREER award (K.K.).", revision_no = "29", abstract = "We report the effects of 5,5,5-trifluoroleucine (TFL) stereochemistry on coiled-coil peptide biosynthesis and stability. We demonstrate that two diastereoisomers of TFL are activated and incorporated into peptides expressed in E. coli. Coiled-coil homodimers of these peptides exhibited increased stability. An equimolar mixture of the two fluorinated peptides formed a heterodimer of modestly enhanced thermal stability relative to the homodimers.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/12672, title ="Two-color labeling of temporally defined protein populations in mammalian cells", author = "Beatty, Kimberly E. and Tirrell, David A.", journal = "Bioorganic and Medicinal Chemistry Letters", volume = "18", number = "22", pages = "5995-5999", month = "November", year = "2008", doi = "10.1016/j.bmcl.2008.08.046", issn = "0960-894X", url = "https://resolver.caltech.edu/CaltechAUTHORS:BEAbmcl08", note = "© 2008 Elsevier. \n\nReceived 12 July 2008; revised 11 August 2008; accepted 12 August 2008. Available online 19 August 2008. \n\nWe thank S.E. Fraser and C. Waters (of the Biological Imaging Center of the Beckman Institute at Caltech) for advice on microscopy. We thank L. Brown and A. Spalla (City of Hope) and R. Diamond and D. Perez (Caltech) for assistance with flow cytometry, and M. Shahgholi for help with mass spectrometry. We appreciate the insightful comments on this work provided by M.L. Mock, Y.Y. Lu, and M.J. Hangauer. This work was supported by NIH Grant GM62523, by the ARO-sponsored Institute for Collaborative Biotechnologies, and by the Joseph J. Jacobs Institute for Molecular Engineering for Medicine. K.E.B. is grateful to the Hertz Foundation, PEO, and the AAAS (Alan E. Leviton Award) for supporting her research. \n\nSupplementary data: Experimental protocols and additional data. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bmcl.2008.08.046.", revision_no = "26", abstract = "The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in fixed cells. Reactive lissamine rhodamine (LR), 7-dimethylaminocoumarin (DMAC), and bodipy-630 (BDPY) dyes were prepared and examined for use in selective dye-labeling of newly synthesized proteins in Rat-1 fibroblasts. The LR and DMAC, but not BDPY, fluorophores were found to enable selective, efficient labeling of subsets of the proteome; cells labeled with Aha and Hpg exhibited fluorescence emission three- to sevenfold more intense than that of control cells treated with Met. We also examined simultaneous and sequential pulse-labeling of cells with Aha and Hpg. After pulse-labeling, cells were treated with reactive LR and DMAC dyes, and labeled cells were imaged by fluorescence microscopy and analyzed by flow cytometry. The results of these studies demonstrate that amino acid labeling can be used to achieve selective two-color imaging of temporally defined protein populations in mammalian cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/14115, title ="Cell response to RGD density in cross-linked artificial extracellular matrix protein films", author = "Liu, Julie C. and Tirrell, David A.", journal = "Biomacromolecules", volume = "9", number = "11", pages = "2984-2988", month = "November", year = "2008", doi = "10.1021/bm800469j", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20090430-093343619", note = "© 2008 American Chemical Society. \n\nReceived April 29, 2008; Revised Manuscript Received August 18, 2008. \n\nWe thank Paul Nowatzki for performing the AFM study, Elizabeth Jones and David Koos for advice on cell migration studies, Marissa Mock for helpful discussions on spin-coating protein films, Scott Fraser for help with fluorescence microscopy, and the Molecular Materials Research Center of the Caltech Beckman Institute for help with XPS. This work was supported by a Whitaker graduate fellowship to J.C.L., by NIH grant EB1971, and by the NSF Center for the Science and Engineering of Materials at the California Institute of Technology.", revision_no = "32", abstract = "This study examines the adhesion, spreading, and migration of human umbilical vein endothelial cells on cross-linked films of artificial extracellular matrix (aECM) proteins. The aECM proteins described here were designed for application in small-diameter grafts and are composed of elastin-like structural repeats and fibronectin cell-binding domains. aECM-RGD contains the RGD sequence derived from fibronectin; the negative control protein aECM-RDG contains a scrambled cell-binding domain. The covalent attachment of poly(ethylene glycol) (PEG) to aECM substrates reduced nonspecific cell adhesion to aECM-RDG-PEG but did not preclude sequence-specific adhesion of endothelial cells to aECM-RGD-PEG. Variation in ligand density was accomplished by the mixing of aECM-RGD-PEG and aECM-RDG-PEG prior to cross-linking. Increasing the density of RGD domains in cross-linked films resulted in more robust cell adhesion and spreading but did not affect cell migration speed. Control of cell-binding domain density in aECM proteins can thus be used to modulate cell adhesion and spreading and will serve as an important design tool as these materials are further developed for use in surgery, tissue engineering, and regenerative medicine.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53231, title ="Unnatural Amino Acid Incorporation into Virus-Like Particles", author = "Strable, Erica and Prasuhn, Duane E.", journal = "Bioconjugate Chemistry", volume = "19", number = "4", pages = "866-875", month = "April", year = "2008", doi = "10.1021/bc700390r", issn = "1043-1802", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150107-082245467", note = "© 2008 American Chemical Society\n\nReceived October 21, 2007;\nRevised Manuscript Received January 14, 2008\n\nThis work was supported by the NIH (AI056013, RR021886, GM62523), the David & Lucille Packard Foundation Interdisciplinary Science Program, and the Canadian Institutes of Health Research (postdoctoral fellowship to A.K.U.). Cryo-electron microscopy was performed at the National Resource for Automated Molecular Microscopy which is supported by the NIH NCRR P41 program (RR17573).", revision_no = "19", abstract = "Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qβ capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qβ suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53575, title ="Mechanically Tunable Thin Films of Photosensitive Artificial Proteins:\u2009 Preparation and Characterization by Nanoindentation", author = "Nowatzki, Paul J. and Franck, Christian", journal = "Macromolecules", volume = "41", number = "5", pages = "1839-1845", month = "March", year = "2008", doi = "10.1021/ma071717a", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150112-113738681", note = "© 2008 American Chemical Society.\nReceived July 31, 2007. Revised Manuscript Received December 12, 2007. Published on Web 02/15/2008. Published In Issue March 11, 2008.\n\nWe gratefully acknowledge support of this research by the Center for the Science and Engineering of Materials at Caltech (NSF DMR-0520565) and by NIH grant\nEB1971. S.A.M. is supported by a pre-doctoral fellowship from\nthe NIH. We thank Marissa Mock for NMR characterization\nand Doron Shilo for help with AFM measurements.", revision_no = "10", abstract = "Thin films of controlled elastic modulus were made by photo-cross-linking artificial extracellular matrix (aECM) proteins containing the photosensitive amino acid p-azidophenylalanine (pN_3Phe). The elastic moduli of the films were calculated from nanoindentation data collected by atomic force microscopy (AFM) using a thin-film Hertz model. The modulus was shown to be tunable in the range 0.3−1.0 MPa either by controlling the irradiation time or by varying the level of pN_3Phe in the protein. Tensile measurements on bulk films of the same proteins and finite-element simulation of the indentation process agreed with the thin-film modulus measurements from AFM. Substrates characterized by spatial variation in elastic modulus were created by local control of the irradiation time.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53576, title ="Enzymatic N-terminal Addition of Noncanonical Amino Acids to Peptides and Proteins", author = "Connor, Rebecca E. and Piatkov, Konstantin", journal = "ChemBioChem", volume = "9", number = "3", pages = "366-369", month = "February", year = "2008", doi = "10.1002/cbic.200700605", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150112-114337627", note = "© 2008 WILEY-VCH Verlag GmbH & Co.\nReceived: October 10, 2007. Published online on January 18, 2008.\n\nThis work was supported by NIH grant GM62523 (to D.A.T.), NIH grant DK39520 (to A.V.), the ARO-sponsored Institute for Collaborative Biotechnologies, and the Beckman Institute at Caltech. We thank Christina Smolke for use of the Molecular Imager and Mona Shahgholi for help with mass spectrometry. Pin Wang and A. James Link kindly provided the purified synthetases. Edman degradation was performed by the Peptide and Protein Molecular Analysis Laboratory of the Beckman Institute at Caltech.", revision_no = "13", abstract = "A chromatographic assay was used to identify ten noncanonical amino acids that can be appended to the N terminus of peptides and proteins by the L,F-transferase of Escherichia coli. A model protein substrate, E. coli dihydrofolate reductase, was modified with p-ethynylphenylalanine and conjugated to azide–biotin and azide–polyethylene glycol–fluorescein probes (see scheme).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53689, title ="Processing of N-Terminal Unnatural Amino Acids in Recombinant Human Interferon-β in Escherichia coli", author = "Wang, Aijun and Nairn, Natalie Winblade", journal = "ChemBioChem", volume = "9", number = "2", pages = "324-330", month = "January", year = "2008", doi = "10.1002/cbic.200700379", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-100617886", note = "c2008 Wiley-VCH Verlag.\n Issue published online: 17 JAN 2008.\n Article first published online: 20 DEC 2007.\n Manuscript Received: 10 JUL 2007.\n\nWe would like to thank Inchan Kwon, Tom Graddis, Kurt Shanebeck, Scott Bloom, and J. Myron Crawford for their advice and assistance. Research on UAAs at Caltech is supported by NIH Grant GM62523.", revision_no = "13", abstract = "Incorporation of unnatural amino acids into recombinant proteins represents a powerful tool for protein engineering and protein therapeutic development. While the processing of the N-terminal methionine (Met) residues in proteins is well studied, the processing of unnatural amino acids used for replacing the N-terminal Met remains largely unknown. Here we report the effects of the penultimate residue (the residue after the initiator Met) on the processing of two unnatural amino acids, L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG), at the N terminus of recombinant human interferon-β in E. coli. We have identified specific amino acids at the penultimate position that can be used to efficiently retain or remove N-terminal AHA or HPG. Retention of N-terminal AHA or HPG can be achieved by choosing amino acids with large side chains (such as Gln, Glu, and His) at the penultimate position, while Ala can be selected for the removal of N-terminal AHA or HPG. Incomplete processing of N-terminal AHA and HPG (in terms of both deformylation and cleavage) was observed with Gly or Ser at the penultimate position.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/11869, title ="Concluding remarks: The importance of polymer science for biological systems", author = "Tirrell, David", journal = "Faraday Discussions", volume = "139", pages = "419-420", month = "January", year = "2008", issn = "1359-6640", url = "https://resolver.caltech.edu/CaltechAUTHORS:TIRfd08", note = "This journal is © The Royal Society of Chemistry 2008. \n\nReceived 22nd May 2008, Accepted 10th June 2008. First published on the web 23rd July 2008.", revision_no = "12", abstract = "The 139th Faraday Discussion covered many interesting subjects, divided into four sections: Cell Interactions; Membranes & Walls; Proteins and Polysaccharides; and Natural & Synthetic Polymers. Here, the meeting is summarised and future prospects are explored.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53697, title ="Site-Specific Incorporation of Tryptophan Analogues into Recombinant Proteins in Bacterial Cells", author = "Kwon, Inchan and Tirrell, David A.", journal = "Journal of the American Chemical Society", volume = "129", number = "34", pages = "10431-10437", month = "August", year = "2007", doi = "10.1021/ja071773r", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-104001124", note = "Copyright © 2007 American Chemical Society.\n Published In Issue August 29, 2007.\n Received March 13, 2007. \n\nWe thank I. C. Tanrikulu, R. Connor, and\nT. H. Yoo for valuable discussions and Dr. J. Zhou for help\nwith LC-MS analysis. This work was supported by National\nInstitutes of Health grant GM62523.", revision_no = "10", abstract = "A designed yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) activates four tryptophan (Trp) analogues (6-chlorotryptophan (6ClW), 6-bromotryptophan (6BrW), 5-bromotryptophan (5BrW), and benzothienylalanine (BT)) that are not utilized by the endogenous E. coli translational apparatus. Introduction of yPheRS (T415G) and a mutant yeast phenylalanine amber suppressor tRNA (ytRNA^(Phe)_(CUA_UG)) into an E. coli expression host allowed site-specific incorporation of three of these analogues (6ClW, 6BrW, and BT) into recombinant murine dihydrofolate reductase in response to amber stop codons with at least 98% fidelity. All three analogues were introduced at the Trp66 position in the chromophore of a cyan fluorescent protein variant (CFP6) to investigate the attendant changes in spectral properties. Each of the analogues caused blue shifts in the fluorescence emission and absorption maxima. The CFP6 variant bearing BT at position 66 exhibited an unusually large Stokes shift (56 nm). An expanded set of genetically encoded Trp analogues should enable the design of new proteins with novel spectral properties.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/9676, title ="Evolution of a fluorinated green fluorescent protein", author = "Yoo, Tae-Hyeon and Link, A. James", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "104", number = "35", pages = "13887-13890", month = "August", year = "2007", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:YOOpnas07", note = "© 2007 by the National Academy of Sciences. \n\nEdited by David H. Baker, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 17, 2007 (received for review March 5, 2007). Published online on August 23, 2007, 10.1073/pnas.0701904104 \n\nWe thank Jin K. Montclare and Frances H. Arnold for helpful discussion. We also thank Mona Shahgholi for assistance with MALDI-MS. This work was supported by National Institutes of Health Grant GM62523 and Office of Naval Research Grant N00014-03-1-0793 (to D.A.T) and by a Samsung Scholarship (to T.H.Y.). \n\nAuthor contributions: T.H.Y. and D.A.T. designed research; T.H.Y. performed research; T.H.Y. and D.A.T. analyzed data; and T.H.Y., A.J.L., and D.A.T. wrote the paper. \n\nThe authors declare no conflict of interest. \n\nThis article is a PNAS Direct Submission. \n\nThis article contains supporting information online at www.pnas.org/cgi/content/full/0701904104/DC1.", revision_no = "27", abstract = "The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved {approx}650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53607, title ="Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer", author = "Link, A. James and Vink, Mandy K. S.", journal = "Nature Protocols", volume = "2", number = "8", pages = "1879-1883", month = "July", year = "2007", doi = "10.1038/nprot.2007.268", issn = "1754-2189", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150113-083748968", note = "© 2007 Nature Publishing Group.\nPublished online 26 July 2007.\n\nWe thank Rebecca Connor and Nick Fisk for refinements to this protocol. This work was supported by NIH, by an NSF graduate fellowship to A.J.L. and by a grant from the Netherlands Organization for Scientific Research to M.K.S.V.", revision_no = "14", abstract = "The azide functional group has assumed a prominent role in chemical biology efforts in recent years. Azides may be readily introduced into proteins upon replacement of methionine residues with the non-canonical amino acid azidohomoalanine (AHA). This protocol describes a synthetic route to AHA based on the copper-catalyzed conversion of amines to azides. An alternate protocol for the preparation of AHA is presented in a companion paper. The synthesis and purification of AHA via the route described herein can be completed in 3–4 days.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53604, title ="Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor", author = "Link, A. James and Vink, Mandy K. S.", journal = "Nature Protocols", volume = "2", number = "8", pages = "1884-1887", month = "July", year = "2007", doi = "10.1038/nprot.2007.269", issn = "1754-2189", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150113-082729873", note = "\n\nThis work was supported by NIH, by an NSF graduate fellowship to A.J.L. and by a grant from the Netherlands Organization for Scientific Research to M.K.S.V.", revision_no = "8", abstract = "This protocol describes a synthetic route to the non-canonical amino acid azidohomoalanine (AHA) using protected homoserine as a starting material. An alternative route to AHA is presented in a companion paper. This synthesis can be completed in 5 days.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53238, title ="High-Throughput Screening for Methionyl-tRNA Synthetases That Enable Residue-Specific Incorporation of Noncanonical Amino Acids into Recombinant Proteins in Bacterial Cells", author = "Yoo, Tae Hyeon and Tirrell, David A.", journal = "Angewandte Chemie International Edition", volume = "46", number = "28", pages = "5340-5343", month = "July", year = "2007", doi = "10.1002/anie.200700779", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-111859005", note = "© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim\n\nReceived: February 20, 2007. Published online: June 14, 2007. \n\nWe thank A. James Link, Kimberly Beatty, and James Van Deventer for helpful discussions. Knocking out of the metE gene from E. coli strain DH10B was done with James Van Deventer. We thank Dr. Mona Shahgholi for assistance with the MALDI-MS and LC-MS analysis. This work was supported by NIH grant GM62523, ONR grant N00014-03-1-0793, and a Samsung Scholarship (to T.H.Y.).", revision_no = "17", abstract = "Aminoacyl-tRNA synthetases (aaRS) with altered substrate specificities have been used to enable both site-specific and residue-specific incorporation of noncanonical amino acids into recombinant proteins. Rational, computational, and combinatorial methods have been employed to engineer the amino acid binding pockets of several aaRS. Combinatorial strategies have been especially effective; Schultz and co-workers have developed powerful methods for selecting aaRS for site-specific incorporation, and we have reported an efficient screening system for use in the global replacement of amino acids. However, because the latter method relies on bio-orthogonal derivatization of noncanonical amino acid side chains, a new approach is needed for the more general problem of activating noncanonical substrates that lack reactive functionality in the side chains. Here we describe a high-throughput method for screening aaRS libraries for the global incorporation of noncanonical amino acids. We demonstrate this strategy by identifying an Escherichia coli methionyl-tRNA synthetase (MetRS) variant that activates 6,6,6-trifluoronorleucine (Tfn, 1; Scheme 1). Tfn does not support significant protein synthesis in conventional E. coli expression strains.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53241, title ="Three-dimensional Full-field Measurements of Large Deformations in Soft Materials Using Confocal Microscopy and Digital Volume Correlation", author = "Franck, C. and Hong, S.", journal = "Experimental Mechanics", volume = "47", number = "3", pages = "427-438", month = "June", year = "2007", doi = "10.1007/s11340-007-9037-9", issn = "0014-4851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-113206162", note = "© Society for Experimental Mechanics 2007\n\nReceived: 27 October 2006 / Accepted: 16 January 2007 / Published online: 8 March 2007\n\nWe gratefully acknowledge the support provided by the National Science Foundation (DMR # 0520565) through the Center for Science and Engineering of Materials (CSEM) at the California Institute of Technology. GR acknowledges the support of the Army Research Office for providing the DURIP funds for the acquisition of the confocal microscope used in this study. GR also gratefully acknowledges the Ronald and Maxine Linde Venture Fund for enabling the acquisition of imaging instrumentation used in this investigation. We would like to thank Mr. Petros Arakelian for his valuable help with the experimental setup.", revision_no = "12", abstract = "A three-dimensional (3-D) full-field measurement technique was developed for measuring large deformations in optically transparent soft materials. The technique utilizes a digital volume correlation (DVC) algorithm to track motions of subvolumes within 3-D images obtained using fluorescence confocal microscopy. In order to extend the strain measurement capability to the large deformation regime (>5%), a stretch-correlation algorithm was developed and implemented into the Fast Fourier Transform (FFT)-based DVC algorithm. The stretch-correlation algorithm uses a logarithmic coordinate transformation to convert the stretch correlation problem into a translational correlation problem under the assumption of small rotation and shear. Estimates of the measurement precision are provided by stationary and translation tests. The proposed measurement technique was used to measure large deformations in a transparent agarose gel sample embedded with fluorescent particles under uniaxial compression. The technique was also employed to measure non-uniform deformation fields near a hard spherical inclusion under far-field uniaxial compression. Introduction of the stretch-correlation algorithm greatly improved the strain measurement accuracy by providing better precision especially under large deformation. Also, the deconvolution of confocal images improved the accuracy of the measurement in the direction of the optical axis. These results shows that the proposed technique is well-suited for investigating cell-matrix mechanical interactions as well as for obtaining local constitutive properties of soft biological materials including tissues in 3-D.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53698, title ="Lithographic Patterning of Photoreactive Cell-Adhesive Proteins", author = "Carrico, Isaac S. and Maskarinec, Stacey A.", journal = "Journal of the American Chemical Society", volume = "129", number = "16", pages = "4874-4785", month = "April", year = "2007", doi = "10.1021/ja070200b", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-104001187", note = "Copyright © 2007 American Chemical Society.\nReceived January 10, 2007 . Publication Date (Web): March 31, 2007. Published In Issue April 25, 2007.\n\nWe thank Michael Diehl, Alireza Ghaffari, and Nandita Sharma for helpful discussion, and Kechun Zhang for help in preparing the patterned substrates. Supported by the NSF\nCenter for the Science and Engineering of Materials at Caltech, NIH GM62523 and EB01971, an NIH predoctoral fellowship to S.A.M., a Whitaker Graduate Fellowship to J.C.L., and the Joseph J. Jacobs Institute for Molecular Engineering for Medicine.", revision_no = "19", abstract = "We describe a novel, simple method for the photolithographic patterning of cell-adhesive proteins. Intrinsically photoreactive proteins are synthesized in Escherichia coli through incorporation of the non-canonical, photosensitive amino acid para-azidophenylalanine. Upon ultraviolet irradiation at 365 nm, proteins form cross-linked films with elastic moduli that can be tuned by varying the concentration of photoreactive amino acid in the expression medium. Films of these proteins can be directly patterned using standard photolithographic techniques. We demonstrate the utility of this method of protein patterning by creating stable arrays of fibroblast cells on an engineered protein “photoresist”. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53608, title ="Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging", author = "Dieterich, Daniela C. and Lee, Jennifer J.", journal = "Nature Protocols", volume = "2", number = "3", pages = "532-540", month = "March", year = "2007", doi = "10.1038/nprot.2007.52", issn = "1754-2189", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150113-084735036", note = "c2007 Nature Publishing Group.\nPublished online 15 March 2007.\n\nWe thank S.A. Kim and E.H. Friedrich for critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute, the Beckman Institute at the California Institute of Technology and NIH (R21DA020589 to E.M.S.). MS analysis was performed at the MS facility of the laboratory of R.J. Deshaies (Howard Hughes Medical Institute, Caltech), which is supported by the Beckman Institute at Caltech and a grant from the Department of Energy to R.J.D. and Barbara J. Wold. D.C.D. is supported by the German Academy for Natural Scientists Leopoldina (BMBF-LPD9901/8-95). J.G. was supported by R.J. Deshaies through Howard Hughes Medical Institute funds. A.J.L. was supported by a National Science Foundation Graduate Research Fellowship.", revision_no = "8", abstract = "A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53242, title ="Non‐Canonical Amino Acids in Protein Polymer Design", author = "Connor, Rebecca E. and Tirrell, David A.", journal = "Journal of Macromolecular Science, Part C: Polymer Reviews", volume = "47", number = "1", pages = "9-28", month = "February", year = "2007", doi = "10.1080/15583720601109552", issn = "1558-3724", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-132717865", note = "Copyright © Taylor & Francis Group, LLC\n\nReceived 2 October 2006; Accepted 31 October 2006.\n\nWork on non-canonical amino acids at Caltech is supported by NIH grants GM62523 and EB1971, by the NSF Center for Science and Engineering of Materials, by the ARO Institute for Collaborative Biotechnologies, and by the Joseph J. Jacobs Institute for Molecular Engineering for Medicine.", revision_no = "10", abstract = "Protein polymers can be prepared with essentially absolute control of chain length, sequence, and stereochemistry through biological synthesis in microbial hosts. But for the polymer chemist, the twenty “canonical” amino acids provide an unacceptably small set of starting materials for macromolecular design. In the past several years, powerful techniques have been developed to introduce non-natural (non-canonical) amino acids containing a wide variety of functional groups into recombinant proteins. This review presents the methods currently available for the introduction of non-natural amino acids into engineered proteins both in vitro and in vivo. Methods for multiple- and single-site introduction are described. Recent applications of these methods are also addressed, particularly the development of biosensors, novel surfaces, and materials.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53243, title ="Dynamic Properties of Artificial Protein Hydrogels Assembled through Aggregation of Leucine Zipper Peptide Domains", author = "Shen, Wei and Kornfield, Julia A.", journal = "Macromolecules", volume = "40", number = "3", pages = "689-692", month = "January", year = "2007", doi = "10.1021/ma0615194", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-134355362", note = "© 2007 American Chemical Society\n\nReceived July 6, 2006; Revised Manuscript Received November 21, 2006\n\nThe authors acknowledge the NSF Center for the Science and Engineering of Materials for financial support.", revision_no = "15", abstract = "Network relaxation dynamics of hydrogels formed from a genetically engineered multidomain protein (AC_(10)A, where A is an associative leucine zipper domain and C_(10) is a random-coil polyelectrolyte domain) were investigated by shear rheometry. Physical gels form by tetrameric association of the leucine zipper end-blocks (A). The longest stress relaxation time (τ_r) of these gels varies strongly with pH, increasing from τ_r ≈ 80 s at pH 8.0 to τ_r ≈ 1000 s at pH 7.0. The rate of strand exchange of the end-blocks was studied by using fluorescence quenching of the labeled form of the A domain. Fluorescence is quenched in solutions of fluorescein-labeled A; dequenching occurs when labeled A is mixed with a 60-fold excess of the unlabeled peptide. The dequenching transient after mixing reveals the characteristic strand exchange time (τ_e) of the A domain. As pH decreases from 8.0 to 7.0, τ_e increases from ca. 200 s to ca. 4500 s. Thus, τ_r of AC_(10)A hydrogels and τ_e of the A domain vary in parallel with pH. The strong correlation between macroscopic and molecular properties indicates that network relaxation is regulated by the lifetime of associations in the transient network. Because the rate of leucine zipper strand exchange is sensitive to interstrand electrostatic interactions, the relaxation behavior of artificial protein hydrogels can be engineered systematically by genetic programming of the amino acid sequence.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/8568, title ="Structure and mechanical properties of artificial protein hydrogels assembled through aggregation of leucine zipper peptide domains", author = "Shen, Wei and Kornfield, Julia A.", journal = "Soft Matter", volume = "3", number = "1", pages = "99-107", month = "January", year = "2007", issn = "1744-6848", url = "https://resolver.caltech.edu/CaltechAUTHORS:SHEsm07", note = "This journal is © The Royal Society of Chemistry 2007. \n\nReceived 31st July 2006, Accepted 20th October 2006. First published on the web 9th November 2006. \n\nThe authors acknowledge the NSF Center for the Science and Engineering of Materials for financial support.", revision_no = "15", abstract = "Artificial protein hydrogels made from a triblock protein (designated AC10A, where A is an acidic zipper domain and C10 comprises 10 repeats of the nonapeptide sequence exhibit normalized plateau storage moduli (G/nkT) less than 0.13 at all concentrations, pH values, and ionic strengths examined. These gels are surprisingly soft due to loop formation at the expense of bridges between physical junctions. Molecular-level evidence of loop formation is provided by strong fluorescence energy transfer (FRET) between distinct chromophores placed at the C- and N-termini of labelled chains diluted in an excess of unlabelled chains. The tendency to form loops originates from the compact size of the random coil midblock (mean RH(C10) 20 Å, determined from quasi-elastic light scattering of C10), and is facilitated by the ability of the leucine zipper domains to form antiparallel aggregates. Although the aggregation number of the leucine zipper domains is small (tetrameric, determined from multi-angle static light scattering of AC10 diblock), the average center-to-center distance between aggregates is roughly 1.5 times the average end-to-end distance of the C10 domain in a 7% w/v network. To avoid stretching the C10 domain, the chains tend to form loops. Changes in pH or ionic strength that expand the polyelectrolyte midblock favor bridging, leading to greater G as long as leucine zipper endblocks do not dissociate. Understanding of the network structure provided successful design strategies to increase the rigidity of these hydrogels. In contrast to intuitive design concepts for rubber and gel materials, it was shown that increasing either the length or the charge density of the midblock increases rigidity, because fewer chains are wasted in loop formation.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53699, title ="Design of a Bacterial Host for Site-Specific Incorporation of p-Bromophenylalanine into Recombinant Proteins", author = "Kwon, Inchan and Wang, Pin", journal = "Journal of the American Chemical Society", volume = "128", number = "36", pages = "11778-11783", month = "September", year = "2006", doi = "10.1021/ja0626281", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-104001262", note = "Copyright © 2006 American Chemical Society.\nPublished In Issue September 13, 2006.\nReceived April 14, 2006.\n\nWe thank Dr. P. Plateau for providing the\nplasmid pXLysKS1 and Dr. M. Shahgholi for assistance with\nmass spectrometry. We are grateful to Dr. K. Shin, Dr. J.\nPetersson, and Dr. D. Beene for help with in vitro transcription of tRNA. We thank I. C. Tanrikulu for help with analysis of the PheRS crystal structure. We also thank Dr. A. J. Link and K. E. Beatty for discussion and valuable comments. This work was supported by National Institutes of Health grant GM62523.", revision_no = "8", abstract = "Introduction of a yeast suppressor tRNA (ytRNA^(Phe)_(CUA)) and a mutant yeast phenylalanyl-tRNA synthetase (yPheRS (T415G)) into an Escherichia coli expression host allows in vivo incorporation of phenylalanine analogues into recombinant proteins in response to amber stop codons. However, high-fidelity incorporation of non-natural amino acids is precluded in this system by mischarging of ytRNA^(Phe)_(CUA) with tryptophan (Trp) and lysine (Lys). Here we show that ytRNA^(Phe)_(CUA) and yPheRS can be redesigned to achieve high-fidelity amber codon suppression through delivery of p-bromophenylalanine (pBrF). Two strategies were used to reduce misincorporation of Trp and Lys. First, Lys misincorporation was eliminated by disruption of a Watson−Crick base pair between nucleotides 30 and 40 in ytRNA^(Phe)_(CUA). Loss of this base pair reduces mischarging by the E. coli lysyl-tRNA synthetase. Second, the binding site of yPheRS was redesigned to enhance specificity for pBrF. Specifically, we used the T415A variant, which exhibits 5-fold higher activity toward pBrF as compared to Trp in ATP−PP_i exchange assays. Combining mutant ytRNA^(Phe)_(CUA) and yPheRS (T415A) allowed incorporation of pBrF into murine dihydrofolate reductase in response to an amber codon with at least 98% fidelity.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53690, title ="Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues", author = "Son, Soojin and Caglar Tanrikulu, I.", journal = "ChemBioChem", volume = "7", number = "8", pages = "1251-1257", month = "August", year = "2006", doi = "10.1002/cbic.200500420", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-100617996", note = "c2006 Wiley-VCH Verlag.\n Issue published online: 27 JUL 2006.\n Article first published online: 7 JUN 2006.\n Manuscript Received: 11 OCT 2005.\n\nWe thank Rich Olson and Pamela Bjorkman for use of and assistance with their analytical ultracentrifuge. We also thank Jin K. Montclare for her help with gel retardation assays and helpful discussions. This work was supported by NIH GM62523.", revision_no = "9", abstract = "Two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and (2S,3R)-4,4,4-trifluorovaline (4TFV), which have been shown to serve as isoleucine surrogates in protein synthesis in Escherichia coli, have been incorporated in vivo into basic leucine zipper (bzip) peptides derived from GCN4. The extents of residue-specific incorporation of 5TFI and 4TFV were 90 and 88\u2009%, respectively, of the encoded isoleucine residues, as evidenced by MALDI mass spectrometry and amino acid analysis. Both circular dichroism and equilibrium sedimentation studies of the fluorinated bzip peptides indicated preservation of secondary and higher-order protein structure. Thermal-denaturation experiments showed an increase of 27\u2009°C in melting temperature when isoleucine was replaced by 5TFI. However, the T_m of the peptide containing 4TFV was increased by only 4\u2009°C over that of the peptide containing valine. Similar trends were observed in chemical denaturation studies in which ΔΔG_(unfold) in water was determined to be 2.1 or 0.3 kcal\u2009mol^(−1) upon incorporation of 5TFI or 4TFV, respectively. When the fluorinated peptides were tested for DNA binding, both their affinity and specificity were similar to those of the respective hydrogenated peptides. These results suggest that fluorinated amino acids, even when introduced into the same positions, can have markedly different effects on the physical properties of proteins, while having little impact on secondary and higher-order structure.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/6880, title ="Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids", author = "Link, A. James and Vink, Mandy K. S.", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "103", number = "27", pages = "10180-10185", month = "July", year = "2006", doi = "10.1073/pnas.0601167103", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:LINpnas06", note = "© 2006 by the National Academy of Sciences \n\nEdited by Dieter Söll, Yale University, New Haven, CT, and approved April 28, 2006 (received for review February 10, 2006). Published online before print June 26, 2006, 10.1073/pnas.0601167103 \n\nWe thank H. Jakubowski (New Jersey Medical School, Newark, NJ) and Y. Mechulam (École Polytechnique, Paliseau, France) for the generous donation of plasmids encoding MetRS. This work was supported by National Institutes of Health Grants GM58867 (to C.R.B.) and GM62523 (to D.A.T.) and by the U.S. Army Research Office through Institute for Collaborative Biotechnologies Grant DAAD19-03-D-0004. \n\nAuthor contributions: A.J.L., C.R.B., and D.A.T. designed research; A.J.L., M.K.S.V., N.J.A., and J.A.P. performed research; A.J.L., M.K.S.V., N.J.A., J.A.P., C.R.B., and D.A.T. analyzed data; and A.J.L. and D.A.T. wrote the paper. \n\nConflict of interest statement: No conflicts declared. \n\nThis paper was submitted directly (Track II) to the PNAS office.", revision_no = "51", abstract = "The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 -> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/5957, title ="Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT)", author = "Dieterich, Daniela C. and Link, A. James", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "103", number = "25", pages = "9482-9487", month = "June", year = "2006", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:DIEpnas06", note = "© 2006 by the National Academy of Sciences \n\nEdited by K. Barry Sharpless, The Scripps Research Institute, La Jolla, CA, and approved May 6, 2006 (received for review February 27, 2006) Published online before print June 12, 2006, 10.1073/pnas.0601637103 \n\nWe thank Dr. Edoardo Marcora (Division of Biology, California Institute of Technology) for the gift of HA-HAP1A; Prof. R.J. Deshaies, Dr. T. Mayor, and all members of the Schuman laboratory for helpful discussions and comments; and Drs. C.-Y. Tai and Y.J. Yoon for critically reading the manuscript. D.C.D. especially thanks Dr. M. Landwehr for many fruitful and critical discussions and S. Materna for the introduction to Python. This work was supported by the Howard Hughes Medical Institute and the Beckman Institute at the California Institute of Technology. MS analysis was performed in the MS facility of the laboratory of R.J. Deshaies (Howard Hughes Medical Institute, California Institute of Technology), which is supported by the Beckman Institute at California Institute of Technology and a grant from the Department of Energy (to R.J. Deshaies) and Barbara J. Wold. D.C.D. is supported by the German Academy for Natural Scientists Leopoldina (Grant BMBF-LPD9901/8-95). J.G. is supported by R. J. Deshaies through Howard Hughes Medical Institute funds. A.J.L. was supported by a National Science Foundation Graduate Research Fellowship. \n\nAuthor contributions: D.C.D., D.A.T., and E.M.S. designed research; D.C.D., A.J.L., and J.G. performed research; D.C.D. and J.G. analyzed data; and D.C.D., D.A.T., and E.M.S. wrote the paper. \n\nConflict of interest statement: No conflicts declared. \n\nThis paper was submitted directly (Track II) to the PNAS office.", revision_no = "8", abstract = "In both normal and pathological states, cells respond rapidly to environmental cues by synthesizing new proteins. The selective identification of a newly synthesized proteome has been hindered by the basic fact that all proteins, new and old, share the same pool of amino acids and thus are chemically indistinguishable. We describe here a technology, based on the cotranslational introduction of azide groups into proteins and the chemoselective tagging of azide-labeled proteins with an alkyne affinity tag, to separate and identify, specifically, the newly synthesized proteins in mammalian cells. Incorporation of the azide-bearing amino acid azidohomoalanine is unbiased, not toxic, and does not increase protein degradation. As a first demonstration of the method, we report the selective purification and identification of 195 metabolically labeled proteins with multidimensional liquid chromatography in-line with tandem MS. Furthermore, in combination with leucine-based mass tagging, candidates were immediately validated as newly synthesized proteins. The identified proteins, synthesized in a 2-h window, possess a broad range of biochemical properties and span most functional gene ontology categories. This technology makes it possible to address the temporal and spatial characteristics of newly synthesized proteomes in any cell type.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/52025, title ="Engineering Cooperativity in Biomotor-Protein Assemblies", author = "Diehl, Michael R. and Zhang, Kechun", journal = "Science", volume = "311", number = "5766", pages = "1468-1471", month = "March", year = "2006", doi = "10.1126/science.1122125 ", issn = "0036-8075", url = "https://resolver.caltech.edu/CaltechAUTHORS:20141120-161852368", note = "© 2006 American Association for the Advancement of Science.\n\n2 November 2005; Accepted 6 February 2006.\n\nWe thank A. J. Link and I. Fushman their help during the\nearly stages of this project; L. Wade and D. Pearson for\nuse of the temperature controller; and P. Wiggins, R. Bao,\nT. Squires, and S. Quake for valuable discussions. This\nwork was supported by the Beckman Foundation through a Beckman Senior Research Fellowship (to M.R.D.) and by a grant from the National Science Foundation.", revision_no = "15", abstract = "A biosynthetic approach was developed to control and probe cooperativity in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds. This approach provides precise control over spatial and elastic coupling between motors. Cooperative interactions between monomeric kinesin-1 motors attached to protein scaffolds enhance hydrolysis activity and microtubule gliding velocity. However, these interactions are not influenced by changes in the elastic properties of the scaffold, distinguishing multimotor transport from that powered by unorganized monomeric motors. These results highlight the role of supramolecular architecture in determining mechanisms of collective transport.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53273, title ="Tuning the erosion rate of artificial protein hydrogels through control of network topology", author = "Shen, Wei and Zhang, Kechun", journal = "Nature Materials", volume = "5", number = "2", pages = "153-158", month = "February", year = "2006", doi = "10.1038/nmat1573", issn = "1476-1122", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-155726855", note = "© 2006 Nature Publishing Group\n\nReceived 23 June 2005; accepted 6 December 2005; published 29 January 2006.\n\nThe authors acknowledge the NSF Center for the Science and Engineering of Materials for financial support. Correspondence and requests for materials should be addressed to J.A.K. or D.A.T. Supplementary Information accompanies this paper on www.nature.com/naturematerials.", revision_no = "20", abstract = "Erosion behaviour governs the use of physical hydrogels in biomedical applications ranging from controlled release to cell encapsulation. Genetically engineered protein hydrogels offer unique means of controlling the erosion rate by engineering their amino acid sequences and network topology. Here, we show that the erosion rate of such materials can be tuned by harnessing selective molecular recognition, discrete aggregation number and orientational discrimination of coiled-coil protein domains. Hydrogels formed from a triblock artificial protein bearing dissimilar helical coiled-coil end domains (P and A) erode more than one hundredfold slower than hydrogels formed from those bearing the same end domains (either P or A). The reduced erosion rate is a consequence of the fact that looped chains are suppressed because P and A tend not to associate with each other. Thus, the erosion rate can be tuned over several orders of magnitude in artificial protein hydrogels, opening the door to diverse biomedical applications.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53691, title ="Stereoselective Incorporation of an Unsaturated Isoleucine Analogue into a Protein Expressed in E. coli", author = "Mock, Marissa L. and Michon, Thierry", journal = "ChemBioChem", volume = "7", number = "1", pages = "83-87", month = "January", year = "2006", doi = "10.1002/cbic.200500201", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-100618078", note = "c2006 Wiley-VCH Verlag.\nIssue published online: 5 JAN 2006. Article first published online: 5 JAN 2006. Manuscript Received: 13 MAY 2005.\n\nThis work was supported by NSF grant DMR0110437, by the Department of Defense through a NDSEG graduate fellowship to\nM.L.M., and by NATO through sabbatical support for T.M. The authors thank Kristi Kiick, Scott Ross, and Takuya Sakaki for their advice and assistance.", revision_no = "13", abstract = "The unsaturated amino acid 2-amino-3-methyl-4-pentenoic acid (E-Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS-E-He and SR-E-Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS-E-Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72%; SR-E-Ile yielded no conclusive evidence for incorporation. ATP/PPi exchange assays indicated that SS-E-Ile was activated by the isoleucyl-tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR-E-Ile was activated approximately 100-times more slowly than SS-E-Ile.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53246, title ="Evolving Proteins of Novel Composition", author = "Montclare, Jin Kim and Tirrell, David A.", journal = "Angewandte Chemie International Edition", volume = "45", number = "27", pages = "4518-4521", month = "January", year = "2006", doi = "10.1002/ange.200600088", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:MONacie2006", note = "© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim\n\nReceived: January 9, 2006\nPublished online: June 8, 2006\n\nWe thank Frances Arnold for use of several instruments. This work was supported by NIH GM 62523 and 5F32 GM67375-2 (D.A.T. and J.K.M.) and by the Beckman Institute of the California Institute of Technology(D.A.T .).", revision_no = "16", abstract = "The behavior of proteins can be altered significantly by the incorporation of noncanonical amino acids. Changes in spectroscopic properties, thermal stability, and molecular recognition behavior have been reported. In many cases, however, introduction of novel amino acids causes loss of protein function. Herein we describe a new approach to protein engineering in which amino acid replacement is combined with directed evolution to create functional proteins of novel composition. Global replacement of the leucine residues of chloramphenicol acetyltransferase (CAT) by 5’,5’,5’-trifluoroleucine (TFL) results in a 20-fold reduction in the half-life (t_(1/2)) of thermal inactivation of the enzyme at 608C. Two rounds of random mutagenesis and screening yielded a variant of CAT containing three amino acid substitutions, which in fluorinated form demonstrates a 27-fold improvement in t_(1/2), recovering the loss in thermostability caused by fluorination.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53224, title ="Fluorescence Visualization of Newly Synthesized Proteins in Mammalian Cells", author = "Beatty, Kimberly E. and Liu, Julie C.", journal = "Angewandte Chemie International Edition", volume = "45", number = "44", pages = "7364-7367", month = "January", year = "2006", doi = "10.1002/anie.200602114", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:BEAacie2006", note = "© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim\n\nReceived: May 26, 2006\nRevised: August 1, 2006\nPublished online: October 11, 2006\n\nWe thank Scott Fraser, Chris Waters, and the Beckman Imaging Center for advice on microscopy, and Rochelle Diamond, Stephanie Adams, and the Caltech Flow Cytometry Facility for assistance with flow cytometry. We thank Anand Asthagiri, Chase Beisel, David Chan, Scott Detmer, Nicholas Graham, Melissa Pope, and Christina Smolke for cell lines and reagents. James Van Deventer made helpful comments on the manuscript. This work was supported by a Fannie and John Hertz Foundation Fellowship (to K.E.B.), a Whitaker Foundation Graduate Fellowship (to J.C.L.), a postdoctoral fellowship from the German Academy for Natural Scientists Leopoldina (BMBF-LPD9901/8-95 to D.C.D.), an NSF-NER grant (to Q.W.), NIH grant GM62523 (to D.A.T.), and the Beckman Institute at Caltech. E.M.S. is an Investigator of the Howard Hughes Medical Institute.", revision_no = "20", abstract = "Modern proteomic methods enable efficient identification of the hundreds or thousands of proteins present in whole cells or in isolated organelles. However, a thorough understanding of the proteome requires insight into protein localization as well as protein identity. Recently, visualization of newly synthesized proteins in bacterial cells was demonstrated through co-translational introduction of an alkynyl amino acid followed by selective CuI-catalyzed ligation of the alkynyl side chain to the fluorogenic dye 3-azido-7-hydroxycoumarin. Here we report that selective fluorescence labeling and imaging of newly synthesized proteins can be accomplished in a diverse set of mammalian cells.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53816, title ="Selective Dye-Labeling of Newly Synthesized Proteins in Bacterial Cells", author = "Beatty, Kimberly E. and Xie, Fang", journal = "Journal of the American Chemical Society", volume = "127", number = "41", pages = "14150-14151", month = "October", year = "2005", doi = "10.1021/ja054643W", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-102630846", note = "© 2005 American Chemical Society. \n\nPublished In Issue October 19, 2005. Publication Date (Web): September 24, 2005. Received July 12, 2005. \n\nAcknowledgment. We thank Christopher W. Waters for help\nwith microscopy, Mona Shahgholi for assistance with mass\nspectrometry, and Christina Smolke for use of her transilluminator.\nWe are grateful to A. James Link for gifts of 4 and pQE30-Barstar,\nand to Marissa Mock and Sanne Schoffelen for advice and\nassistance. This work was supported by a Fannie and John Hertz\nFoundation Fellowship to K.E.B., by the National Institutes of\nHealth, and by the Beckman Institute at Caltech. \n\nSupporting Information Available: Experimental protocols.", revision_no = "11", abstract = "We describe fluorescence labeling of newly synthesized proteins in Escherichia coli cells by means of Cu(I)-catalyzed cycloaddition between alkynyl amino acid side chains and the fluorogenic dye 3-azido-7-hydroxycoumarin. The method involves co-translational labeling of proteins by the non-natural amino acids homopropargylglycine (Hpg) or ethynylphenylalanine (Eth) followed by treatment with the dye. As a demonstration, the model protein barstar was expressed and treated overnight with Cu(I) and 3-azido-7-hydroxycoumarin. Examination of treated cells by confocal microscopy revealed that strong fluorescence enhancement was observed only for alkynyl-barstar treated with Cu(I) and the reactive dye. The cellular fluorescence was punctate, and gel electrophoresis confirmed that labeled barstar was localized in inclusion bodies. Other proteins showed little fluorescence. Examination of treated cells by fluorimetry demonstrated that cultures supplemented with Eth or Hpg showed an 8- to 14-fold enhancement in fluorescence intensity after labeling. Addition of a protein synthesis inhibitor reduced the emission intensity to levels slightly above background, confirming selective labeling of newly synthesized proteins in the bacterial cell. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53275, title ="Template-Directed Olefin Cross Metathesis", author = "Cantrill, Stuart J. and Grubbs, Robert H.", journal = "Organic Letters", volume = "7", number = "19", pages = "4213-4216", month = "September", year = "2005", issn = "1523-7060", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-125549100", note = "© 2005 American Chemical Society. \n\nReceived July 7, 2005. \n\nThe authors thank the Office of Naval Research (ONR) for its support through the Multidisciplinary University Research Initiative (MURI) program, the Alexander von Humboldt Foundation for a Feodor Lynen postdoctoral fellowship to S.P.S., the Croucher Foundation (HKSAR) for a postdoctoral fellowship to K.C.-F.L., and the National Institutes of Health for a postdoctoral fellowship to A.N.", revision_no = "18", abstract = "A template containing two secondary dialkylammonium ion recognition sites for encirclement by olefin-bearing dibenzo[24]crown-8 derivatives has been used to promote olefin cross metatheses with ruthenium-alkylidene catalysts. For monoolefin monomers, the rates of metatheses and yields of the dimers are both amplified in the presence of the template. Likewise, for a diolefin monomer, the yield of the dimer is enhanced in the presence of the template under conditions where higher oligomers are not formed.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53732, title ="Controlled structure in artificial protein hydrogels", author = "Kennedy, Scott B. and Littrell, Kenneth", journal = "Macromolecules", volume = "38", number = "17", pages = "7470-7475 ", month = "August", year = "2005", doi = "10.1021/ma050726l", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-141719887", note = "Copyright © 2005 American Chemical Society. \n\nReceived April 7, 2005; Revised Manuscript Received June 6, 2005. Publication Date (Web): July 21, 2005. \n\nThis work was supported by the\nMaterials Research Science and Engineering Center at\nthe University of Massachusetts Amherst (DMR-9809365). The authors thank Paul Welch for assistance\nin the modeling and simulation of SAXS data and\nSoenke Seifert in the SAXS measurements. This work\nbenefited from the use of BESSRC-CAT at APS and\nIPNS, funded by the U.S. DOE, BES under Contract\nW-31-109-ENG-38 to the University of Chicago.", revision_no = "11", abstract = "Small-angle X-ray scattering (SAXS) and circular dichroism (CD) were used to study the structure of artificial, multidomain protein hydrogels. Preliminary sedimentation equilibrium results indicate that the flanking leucine zipper domains used in the design of the artificial multidomain protein form tetrameric helical bundles. The leucine zipper domain alone precipitates at high concentrations (7% w/v) and near-neutral pH, but the multidomain protein remains soluble owing to a hydrophilic central domain. The resulting solution displays characteristic properties of physical gels. SAXS data from gels fit well to a cylindrical model with the following dimensions:\u2009 length 63 Å, radius 13.6 Å, and a 1 Å axial pore. These results match the dimensions of a tetrameric helical bundle and indicate that the low concentration equilibrium structure of the leucine zipper domain is maintained within the multidomain protein, even at high concentrations. Altogether, these results confirm a static picture of the gel structure where tetrameric self-associations of leucine zipper domains act as physical cross-links in the protein hydrogel.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53279, title ="Protein engineering approaches to biomaterials design", author = "Maskarinec, Stacey A. and Tirrell, David A.", journal = "Current Opinion in Biotechnology", volume = "16", number = "4", pages = "422-426", month = "August", year = "2005", doi = "10.1016/j.copbio.2005.06.009", issn = "0958-1669", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-123322094", note = "© 2005 Elsevier Ltd. All rights reserved.\n\nThis review comes from a themed issue on Protein technologies and commercial enzymes.\nEdited by Bernhard Hauer and Brian K Kay", revision_no = "10", abstract = "Biomaterials play crucial roles in reconstructive surgery, tissue engineering and regenerative medicine. Protein engineering offers powerful solutions to the challenges posed by the creation of well-defined, multifunctional materials that guide cell and tissue behavior. Especially challenging is the complex interplay between mechanical and biological properties in determining the success or failure of biomaterials designed for clinical use.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53700, title ="Artificial Polypeptide Scaffold for Protein Immobilization", author = "Zhang, Kechun and Diehl, Michael R.", journal = "Journal of the American Chemical Society", volume = "127", number = "29", pages = "10136-10137", month = "July", year = "2005", doi = "10.1021/ja051457h", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-104001317", note = "Copyright © 2005 American Chemical Society.\nPublished In Issue July 27, 2005.\nPublication Date (Web): June 29, 2005.\nReceived March 7, 2005. \n\nThis work was supported by the NSF sponsored\nCenter for the Science and Engineering of Materials at\nthe California Institute of Technology.", revision_no = "11", abstract = "An artificial polypeptide scaffold composed of surface anchor and protein capture domains was designed and expressed in vivo. By using a mutant E. coli phenylalanyl−tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated into the surface anchor domain. Octyltrichlorosilane-treated surfaces were functionalized with this polypeptide by spin coating and photocrosslinking. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53901, title ="Reassignment of sense codons in vivo", author = "Link, A. James and Tirrell, David A.", journal = "Methods", volume = "36", number = "3", pages = "291-298", month = "July", year = "2005", doi = "10.1016/j.ymeth.2005.04.005", issn = "1046-2023", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-160048550", note = "c2005 Elsevier Inc.\nAccepted 28 April 2005.\n\nWork on codon reassignment at Caltech has been supported by grants from the National Science Foundation (DMR 0080065), the National Institutes of Health (GM 62523), the U.S. Army Research Office (DAAD19-03-D-0004), and the Beckman Institute Endowment.", revision_no = "9", abstract = "The genetic code maps one or more of the 61 sense codons onto a set of 20 canonical amino acids. Reassignment of sense codons to non-canonical amino acids in model organisms such as Escherichia coli has been achieved through manipulation of the cellular protein synthesis machinery. Specifically, control of amino acid pools, coupled with engineering of the aminoacyl-tRNA synthetase activity of the host, has enabled assignment of sense codons to a wide variety of non-canonical amino acids under conditions routinely used for expression of recombinant proteins. Codon reassignment is leading to important advances in protein engineering and bioorganic chemistry. Here we summarize some of those advances, and provide detailed protocols for codon reassignment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53711, title ="Molecular Level Investigations of the Inter- and Intramolecular Interactions of pH-Responsive Artificial Triblock Proteins", author = "Stevens, Molly M. and Allen, Stephanie", journal = "Biomacromolecules", volume = "6", number = "3", pages = "1266-1271", month = "May", year = "2005", doi = "10.1021/bm049369x", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-121449462", note = "Copyright © 2005 American Chemical Society.\nPublished In Issue May 09, 2005. Publication Date (Web): March 17, 2005. Received October 6, 2004. Revised Manuscript Received January 28, 2005.\n\nAcknowledgment. The authors thank Prof. H. E. Gaub\n(Ludwig-Maximilians-Universita¨t, Munich) for access to\ncustom-built AFM instrumentation during the early stages\nof this study. Work at Nottingham was supported by the\nBBSRC, and S.A. thanks Pfizer Global Research and\nDevelopment (Sandwich, Kent, UK) for the funding of her\nlectureship. Work at Caltech was supported by the NSF\nCenter for the Science and Engineering of Materials.", revision_no = "8", abstract = "Intelligent materials that can undergo physical gelation in response to environmental stimuli have potential impacts in the bioengineering and biomedical fields where the entrapment of cellular or molecular species is desired. Here, we utilize atomic force microscopy (AFM) to perform molecular level investigations of designer artificial proteins that undergo physical gelation. These are engineered as triblock copolymers with independent interchain binding and solvent retention functions, namely, two terminal leucine zipper-like peptide sequences and a central alanylglycine rich sequence, respectively. AFM force measurements between probes and surfaces functionalized with molecules of this triblock protein revealed adhesive interactions that increased in average force and frequency as the pH was lowered from pH 11.2 to 7.4 to 4.5, reflecting an increase in the numbers of interacting molecular strands. In bulk solution, lowering the pH results in a viscous liquid to gel transition. The modular design of the triblock protein was also exploited for single molecule force spectroscopy investigations, which revealed altered intramolecular interactions in response to changes in pH. An increased understanding of the inter- and intramolecular forces involved in biomolecule driven gelation processes is not only of great fundamental interest in the study of the biomolecular systems involved but may also prove key in enabling the rational design of new generations of intelligent hydrogel systems. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53282, title ="Assembly of an Artificial Protein Hydrogel through Leucine Zipper Aggregation and Disulfide Bond Formation", author = "Shen, Wei and Lammertink, Rob G. H.", journal = "Macromolecules", volume = "38", number = "9", pages = "3909-3916", month = "May", year = "2005", doi = "10.1021/ma048348s", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-120951540", note = "© 2005 American Chemical Society\n\nReceived August 11, 2004; Revised Manuscript Received February 3, 2005\n\nThe authors acknowledge the NSF Center for the Science and Engineering of Materials for financial support.", revision_no = "10", abstract = "We present a strategy to stabilize artificial protein hydrogels through covalent bond formation following physical association of terminal leucine zipper domains. Artificial proteins consisting of two terminal leucine zipper domains and a random coil central domain form transient networks above a certain concentration, but the networks dissolve when placed in excess buffer. Engineering of a cysteine residue into each leucine zipper domain allows formation of disulfide bonds templated by leucine zipper aggregation. Circular dichroism spectra show that the zipper domains remain helical after cysteine residues and disulfide bonds are introduced. Asymmetric placement of the cysteine residues in the leucine zipper domains suppresses intramolecular disulfide bonds and creates linked “multichains” composed of ca. 9 protein chains on average, as determined by multiangle light scattering measurements. These “multichains” act as the building units of the physical network formed by leucine zipper aggregation. The increased valency of the building units stabilizes the hydrogels in open solutions, while the physical nature of their association allows the reversibility of gelation to be retained. The gel networks dissolve at pH 12.2, where the helicity of the leucine zipper domains is reduced by ca. 90%, and re-form upon acidification. The hydrogels show anisotropic swelling when anchored on aminated surfaces and may find applications in tissue engineering, controlled release, and microarray technologies on the basis of their stability, reversibility, and swelling behavior.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53283, title ="Controlled Assembly of Macromolecular β-Sheet Fibrils", author = "Smeenk, Jurgen M. and Otten, Matthijs B. J.", journal = "Angewandte Chemie International Edition", volume = "44", number = "13", pages = "1968-1971", month = "March", year = "2005", doi = "10.1002/anie.200462415", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-092941334", note = "© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim\n\nReceived: October 25, 2004\nPublished online: February 21, 2005\n\nJ.T. gratefully acknowledges the financial support of DSM Research.\nD.A.T. acknowledges National Sciences Foundation grant no. DMR-0110437.", revision_no = "16", abstract = "Construction of functional molecular devices by directed assembly processes is one of the main challenges in the field of nanotechnology. Many approaches to this challenge use biological assembly as a source of inspiration for the build up of new materials with controlled organization at the nanoscale. In particular, the self-assembly properties of β-sheet peptides have been used in the design of supramolecular materials, such as tapes, nanotubes, and fibrils.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53712, title ="Cell-Binding Domain Context Affects Cell Behavior on Engineered Proteins", author = "Heilshorn, Sarah C. and Liu, Julie C.", journal = "Biomacromolecules", volume = "6", number = "1", pages = "318-323", month = "January", year = "2005", doi = "10.1021/bm049627q", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-121449592", note = "Copyright © 2005 American Chemical Society.\nPublished In Issue January 10, 2005. Publication Date (Web): December 4, 2004. Received June 29, 2004. Revised Manuscript Received October 5, 2004.\n\nAcknowledgment. This work was supported by NIH Grant 5 R01 HL59987-03, NSF Grant BES-9901648, and a Whitaker graduate fellowship. We thank Kathleen Di Zio for helpful discussion regarding protein purification, Paul Nowatzki for providing protein samples, the Electron Microscopy Laboratory in the Biology Division at Caltech and Robert Strittmatter for help with the scanning electron micrographs, and Krystle Wang, Gustavo Olm, and Regina Wilpiszeski for help with the BCA and cell detachment experiments.\n\nSupporting Information Available. Supporting figures\ninclude 1. Cell resistance to detachment forces on engineered\nproteins adsorbed to glass substrates, 2. Western analysis\nconfirming complete cleavage of heptahistidine- and T7-tags,\nand 3. Cell resistance to detachment forces on engineered\nproteins with heptahistidine- and T7-tags removed.", revision_no = "11", abstract = "A family of artificial extracellular matrix proteins developed for application in small-diameter vascular grafts is used to examine the importance of cell-binding domain context on cell adhesion and spreading. The engineered protein sequences are derived from the naturally occurring extracellular matrix proteins elastin and fibronectin. While each engineered protein contains identical CS5 cell-binding domain sequences, the lysine residues that serve as cross-linking sites are either (i) within the elastin cassettes or (ii) confined to the ends of the protein. Endothelial cells adhere specifically to the CS5 sequence in both of these proteins, but cell adhesion and spreading are more robust on proteins in which the lysine residues are confined to the terminal regions of the chain. These results may be due to altered protein conformations that affect either the accessibility of the CS5 sequence or its affinity for the α_4β_1 integrin receptor on the endothelial cell surface. Amino acid choice outside the cell-binding domain can thus have a significant impact on the behavior of cells cultured on artificial extracellular matrix proteins.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53903, title ="Quantitatively distinct requirements for signaling-competent cell spreading on engineered versus natural adhesion ligands", author = "Richman, Gabriel P. and Tirrell, David A.", journal = "Journal of Controlled Release", volume = "101", number = "1-3", pages = "3-12", month = "January", year = "2005", doi = "10.1016/j.jconrel.2004.07.034", issn = "0168-3659", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-160852739", note = "c2004 Elsevier B.V.\nReceived 24 June 2004; accepted 7 July 2004; Available online 12 October 2004.\n\nThe authors thank members of the A.R.A. and\nD.A.T. labs for helpful discussions and technical\nassistance. This work was funded by a start-up grant\nfrom California Institute of Technology to A.R.A.\nand NIH grant 5 R01 HL59987-03 to D.A.T. G.P.R.\nwas partially funded by a graduate fellowship from\nthe NSF-sponsored Materials Research Science and\nEngineering Center at California Institute of\nTechnology.", revision_no = "9", abstract = "To design synthetic microenvironments that elicit desired cell behaviors, we must better understand the molecular mechanisms by which cells interact with candidate biomaterials. Using cell lines with distinct α5β1 integrin expression profiles, we demonstrate that this integrin mediates cell spreading on substrata coated with genetically engineered artificial extracellular matrix (aECM) proteins containing the RGD sequence (RGD-containing aECM protein [^aRGD]) but lacking the PHSRN synergy site. Furthermore, ^aRGD-mediated adhesion stimulates an intracellular focal adhesion kinase (FAK) signal that is indicative of integrin tethering. Although both ^aRGD and the natural ECM protein fibronectin (FN) support α5β1 integrin-mediated cell spreading, quantitative single-cell analysis revealed that aRGD-mediated spreading requires ten-fold greater threshold amount of integrin expression than FN-mediated spreading. Our analysis demonstrates that ^aRGD-based substrata mediate both biophysical (cell spreading) and biochemical (FAK signaling) events via the α5β1 integrin, albeit with efficacy quantitatively distinct from that of natural ECM proteins that possess the full spectrum of adhesion and synergy domains.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53305, title ="Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents", author = "Chen, Tao and McIntosh, Deirdre", journal = "Molecular Membrane Biology", volume = "21", number = "6", pages = "385-393", month = "November", year = "2004", doi = "10.1080/09687860400010516", issn = "0968-7688", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150130-161321146", note = "© 2004 Taylor & Francis Ltd\n\nReceived 11 June 2004; and in revised form 23 August 2004.", revision_no = "15", abstract = "Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C_(10) alkylamines to produce C_(10)-PEAA. Incubation of C_(10)-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C_(10)-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C_(10)-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C_(10)-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the Clo-PEAA-LUV. It is concluded that C_(10)-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53817, title ="Presentation and Detection of Azide Functionality in Bacterial Cell Surface Proteins", author = "Link, A. James and Vink, Mandy K. S.", journal = "Journal of the American Chemical Society", volume = "126", number = "34", pages = "10598-10602", month = "September", year = "2004", doi = "10.1021/ja047629c", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-102630933", note = "Copyright © 2004 American Chemical Society.\nPublished In Issue September 01, 2004. Publication Date (Web): August 3, 2004. Received April 23, 2004.\n\nAcknowledgment. We thank Timothy Chan and Profs. Valery\nFokin and K. Barry Sharpless for their gift of compound 5. We\nare grateful to Han Peeters and Mona Shahgoli for assistance\nwith mass spectrometry. We also thank Prof. Richard Roberts\nfor use of his cell sorter. This work was supported by the ARO\nInstitute for Collaborative Biotechnologies (Grant No. DAAD19-\n03-D-0004), the NSF Center for the Science and Engineering\nof Materials (Grant No. DMR 008 0065), NIH Grant GM62523,\nand the Beckman Institute at the California Institute of Technology.\nA.J.L. is an NSF Graduate Research Fellow. M.K.S.V. was\nsupported by a grant from The Netherlands Organization for\nScientific Research.", revision_no = "8", abstract = "An improved protocol for copper-catalyzed triazole formation on the bacterial cell surface is described. Addition of highly pure CuBr to cells treated with azidohomoalanine (2) leads to ca. 10-fold more extensive cell surface labeling than previously observed. This highly active catalyst allows detection of the methionine analogues azidoalanine (1), azidonorvaline (3), and azidonorleucine (4) in cell surface proteins. Azidoalanine was previously believed to be silent with regard to the cellular protein synthesis machinery. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53904, title ="pH-Dependent Behavior of Surface-immobilized Artificial Leucine Zipper Proteins", author = "Stevens, Molly M. and Allen, Stephanie", journal = "Langmuir", volume = "20", number = "18", pages = "7747-7752", month = "August", year = "2004", doi = "10.1021/la030440e", issn = "0743-7463", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150120-162146672", note = "Copyright © 2004 American Chemical Society.\nPublished In Issue August 31, 2004. Publication Date (Web): August 6, 2004. Received December 29, 2003. Revised June 10, 2004.\n\nAcknowledgment. The authors would like to thank\nScientific and Medical Products Ltd (Cheshire, United\nKingdom) for the kind loan of the Q-sense D300 measurements\nsystem and John Booth of Scientific and Medical\nProducts for his comments and advice in carrying out these experiments. The authors would also like to thank\nPharmacia Biosensor AB (Uppsala, Sweden) for the kind\nloan of the BIAcore 3000 biosensor instrument and Phil\nBuckle of Pharmacia Biosensor AB for his comments and\nadvice in carrying out the experiments. The authors\nthank Dr. Chun Wang for help and advice with the CD\nmeasurements. Work at Nottingham was supported by\nthe BBSRC and S.A. thanks Pfizer Global Research and\nDevelopment (Sandwich, Kent, United Kingdom) for the\nfunding of her lectureship.Workat Caltech was supported\nby the NSF Center for the Science and Engineering of\nMaterials.", revision_no = "8", abstract = "The coiled-coil protein motif occurs in over 200 proteins and has generated interest for a range of applications requiring surface immobilization of the constituent peptides. This paper describes an investigation of the environment-responsive behavior of a monolayer of surface-immobilized artificial proteins, which are known to assemble to form coiled-coil structures in bulk solution. An extended version of the quartz crystal microbalance (QCM-D) and surface plasmon resonance (SPR) are independently employed to characterize the adsorption of the proteins to a gold surface. The data suggest that the molecules arrange in a closely packed layer orientated perpendicular to the surface. QCM-D measurements are also employed to measure pH-induced changes in the resonant frequency (f) and the energy dissipation factor (D) of a gold-coated quartz crystal functionalized with the formed monolayer. Exposure of the protein monolayer to a pH 4.5 solution results in a shift of 43 Hz in f and a shift of −0.7 × 10^(-6) in D as compared to pH 7.4. In contrast, increasing the pH to 11.2, results in f and D shifts of −17 Hz and 0.6 × 10^(-6), respectively. The magnitude of the observed shifts suggests that the proteins form a rigid layer at low pH that can be hydrated to a fluid layer as the pH is increased. These observations correlate with spectroscopic changes that indicate a reduction in the helical content of the protein in bulk solutions of high pH.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53288, title ="Chemical biology: Hitting the sweet spot", author = "Tirrell, David A.", journal = "Nature", volume = "430", number = "7002", pages = "837", month = "August", year = "2004", doi = "10.1038/430837a", issn = "0028-0836", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150121-093748562", note = "© 2004 Nature Publishing Group.", revision_no = "10", abstract = "Delivering chemical agents to specific locations in the body has been an elusive goal ever since Paul Erhlich described his concept of the ‘magic bullet’ nearly a century ago.Whether the objective is delivering drugs to sites of injury and disease, or the infusion of imaging agents that allow physicians to see what is going on, one would like to be able to target molecules to the places where they are needed most. Ideally, one would be able to pick out a single cell (such as a cancer cell) in\nthe midst of many others, and deliver a molecular payload to that cell alone.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53960, title ="Alternative Translations of a Single RNA Message: An Identity Switch of (2S,3R)-4,4,4-Trifluorovaline between Valine and Isoleucine Codons", author = "Wang, Pin and Fichera, Alfio", journal = "Angewandte Chemie International Edition", volume = "43", number = "28", pages = "3664-3666", month = "July", year = "2004", doi = "10.1002/anie.200454036", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150121-144351506", note = "© 2004 Wiley VCH. Issue published online: 7 JUL 2004. Article first published online: 21 JUN 2004. Manuscript Received: 17 FEB 2004.\n\nThis work was supported by the National Institutes of Health (grant nos. GM65500 and GM62523), by a National Sciences Foundation CAREER award (CHE-0236846), and by the National Science Foundation Center for Science and Engineering of Materials at the California Institute of Technology.", revision_no = "14", abstract = "Changed in translation: Bacterial expression hosts can be engineered so that a single RNA message can be read in different ways depending on the relative rates of competing aminoacylation reactions. The (2S,3R)-4,4,4-trifluorovaline can be assigned either to isoleucine or to valine codons according to whether the bacterial host overexpresses the isoleucyl- or the valyl-tRNA synthetase (IleRS and ValRS, respectively; see scheme).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53291, title ="Coiled-Coil Peptide-Based Assembly of Gold Nanoparticles", author = "Stevens, Molly M. and Flynn, Nolan T.", journal = "Advanced Materials", volume = "16", number = "11", pages = "915-918", month = "June", year = "2004", doi = "10.1002/adma.200306430", issn = "0935-9648", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150121-101000800", note = "© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim\n\nReceived: November 6, 2003\nFinal version: January 23, 2004\nPublished online: April 20, 2004\n\nWe thank Professor Robert Sauer for the use of the Aviv spectropolarimeter and Jill Sakata for help with the bacterial expression of the peptides. We acknowledge the support of the National Institutes of Health (Grant #DE-13023).", revision_no = "9", abstract = "The ability to direct the assembly of inorganic nanoparticles using biomolecular recognition is of growing interest in the development of new materials and nanotechnology devices.[1,2] Previous studies have described the use of the highly specific biomolecular recognition systems of DNA,[3,4] the streptavidin/biotin system,[5,6] antibody/antigen systems,[7] and a peptide based linker system[8] to direct nanoparticle assembly. The reversibility of the biological assembly process has not been probed in depth for these nanoparticle systems, with the exception of temperature changes to control the assembly of DNA-linked nanoparticles.[3] For certain applications in medical science such as the generation of novel tunable and/or switchable materials potentially useful for sensing in vivo and drug delivery, the ability to dynamically assemble and disassemble such structures when triggered by physiologically accessible environmental conditions such as changes in pH would be valuable.[1,9] Here we report the coiled-coil-based assembly of gold nanoparticles and demonstrate that the system can be controlled under mild conditions (near-neutral pH and ambient temperature). The flexibility in design afforded\nby varying the peptide sequence to produce coiled coils with different stabilities is also highlighted through the generation of more stable binary nanoparticle systems with controlled spacing and architecture.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53304, title ="Parallel β-Sheet Assemblies at Interfaces", author = "Sneer, Ronit and Weygand, Markus Jan", journal = "ChemPhysChem", volume = "5", number = "5", pages = "747-750", month = "May", year = "2004", doi = "10.1002/cphc.200301046", issn = "1439-4235", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150128-104116321", note = "© 2004 Wiley-VCHV erlag GmbH& Co. KGaA, Weinheim\n\nReceived: November 7, 2003 [Z1046]\n\nThis work was supported by the United States-Israel Binational Science Foundation (Grant No. 2001149) and by the IHP-Contract HPRI-CT-2001±00140 of the European Commission. We acknowledge HASYLAB for synchrotron beamtime. We thank Leslie Leiserowitz for helpful discussions, Inna Solomonov for synchrotron measurements, Mark Karpasas for MALDI measurements, and Vitaly Yerochimovich for help with the FTIR work.", revision_no = "15", abstract = "Polypeptide assemblies may exhibit various topologies[1-9] that are of interest for nanometer-scale surface patterning and its potential applications. The success in designing such ordered molecular architectures entails control over peptide conformations and intermolecular interactions. At the air-water interface peptides composed of alternating hydrophilic and hydrophobic amino acids tend to adopt β-sheet structures[10] yet the repetitive nature of these peptides also promotes nonspecific intermolecular aggregation. Recently, in several systems of de novo designed b-sheet peptides two-dimensional order has been demonstrated by grazing incidence X-ray diffraction[8, 9] and by scanning probe microscopy;[11] the extent of molecular registry has been associated with peptide composition and molecular chain length. Here we aim at formation of parallel β-sheet ordered assemblies at interfaces by using strands programmed to adopt distinct intermolecular electrostatic interactions.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53946, title ="From Giant Vesicles to Filaments and Wires: Templates for Conducting Polymers", author = "Kros, A. and Linhardt, J. G.", journal = "Advanced Materials", volume = "16", number = "8", pages = "723-727", month = "April", year = "2004", doi = "10.1002/adma.200306250", issn = "0935-9648", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150121-114548453", note = "Copyright © 2004 WILEY-VCH Verlag GmbH & Co.\nIssue published online: 26 APR 2004. Article first published online: 26 APR 2004. Manuscript Accepted: 23 DEC 2003. Manuscript Received: 1 OCT 2003.\n\nThis research is financially supported by the Technology Foundation STW, applied science division of NWO and the Technology Programme of the Netherlands Ministry of Economic Affairs and by the NSF Center for the Science and Engineering of Materials at Caltech.", revision_no = "10", abstract = "Conducting polypyrrole-coated structures with lengths of several hundreds of micrometers and diameters varying from several micrometers to less then 150\u2009nm have been formed using biomembrane templates (see Figure). The nanotubes can be attached between Au electrode surfaces, and are stable for at least a month. Possible applications exist in the fabrication of electronic devices, inter-element wiring, and biosensors.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53361, title ="Designing materials for biology and medicine", author = "Langer, Robert and Tirrell, David A.", journal = "Nature", volume = "428", number = "6982", pages = "487-492", month = "April", year = "2004", doi = "10.1038/nature02388", issn = "0028-0836", url = "https://resolver.caltech.edu/CaltechAUTHORS:LANn2004", note = "© 2004 Macmillan Publishers Limited. \n\nThis work was supported in part by the National Institutes of Health. \n\nThe authors declare no competing financial interests.", revision_no = "19", abstract = "Biomaterials have played an enormous role in the success of medical devices and drug delivery systems. We discuss here new challenges and directions in biomaterials research. These include synthetic replacements for biological tissues, designing materials for specific medical applications, and materials for new applications such as diagnostics and array technologies.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/54040, title ="Physical properties of artificial extracellular matrix protein films prepared by isocyanate crosslinking", author = "Nowatzki, Paul J. and Tirrell, David A.", journal = "Biomaterials", volume = "25", number = "7-8", pages = "1261-1267", month = "April", year = "2004", doi = "10.1016/S0142-9612(03)00635-5", issn = "0142-9612", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150123-161424688", note = "© 2003 Elsevier Ltd.\nReceived 4 June 2003; accepted 25 July 2003.\nPublished in issue: Volume 25, Issues 7–8, March–April 2004, Pages 1261–1267.\n\nAcknowledgments: This work was supported by the National Institutes of Health (R01-HL59987) and the National Science Foundation (BES-9901648).", revision_no = "8", abstract = "Artificial extracellular matrix proteins, genetically engineered from elastin- and fibronectin-derived repeating units, were crosslinked with hexamethylene diisocyanate in dimethylsulfoxide. The resulting hydrogel films were transparent, uniform, and highly extensible. Their tensile moduli depended on crosslinker concentration and spanned the range characteristic of native elastin. The water content of the films was low (∼27%), but the temperature-dependent swelling behavior of the crosslinked materials was reminiscent of the lower critical solution temperature property of the soluble polymers.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53713, title ="Comparative Cell Response to Artificial Extracellular Matrix Proteins Containing the RGD and CS5 Cell-Binding Domains", author = "Liu, Julie C. and Heilshorn, Sarah C.", journal = "Biomacromolecules", volume = "5", number = "2", pages = "497-504", month = "March", year = "2004", doi = "10.1021/bm034340z", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-121449681", note = "Copyright © 2004 American Chemical Society.\nPublished In Issue March 08, 2004. Publication Date (Web): January 24, 2004. Received September 4, 2003. Revised Manuscript Received December 8, 2003.\n\nAcknowledgment. We thank Kathleen Di Zio for helpful\ndiscussions regarding the DNA cloning and protein purification, Anand Asthagiri for helpful discussions regarding the cell spreading studies, and Scott Fraser and Carole Lu for help with the fluorescence microscopy. This work was supported by NIH Grant 5 RO1 HL59987-03, NSF Grant\nBES-9901648, and a Whitaker graduate fellowship.", revision_no = "7", abstract = "This study addresses endothelial cell adhesion and spreading on a family of artificial extracellular matrix (aECM) proteins designed for application in small-diameter vascular grafts. The aECM proteins contain domains derived from elastin and from fibronectin. aECM 1 contains the RGD sequence from the tenth type III domain of fibronectin; aECM 3 contains the fibronectin CS5 cell-binding domain. Negative control proteins aECM 2 and 4 are scrambled versions of aECM 1 and 3, respectively. Competitive peptide inhibition studies and comparisons of positive and negative control proteins confirm that adhesion of HUVECs to aECM proteins 1 and 3 is sequence specific. When subjected to a normal detachment force of 780 pN, 3-fold more HUVECs remained adherent to aECM 1 than to aECM 3. HUVECs also spread more rapidly on aECM 1 than on aECM 3. These results (i) indicate that cellular responses to aECM proteins can be modulated through choice of cell-binding domain and (ii) recommend the RGD sequence for applications that require rapid endothelial cell spreading and matrix adhesion. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53310, title ="Artificial Niches for Human Adult Neural Stem Cells: Possibility for Autologous Transplantation Therapy", author = "Liu, Charles Y. and Westerlund, Ulf", journal = "Journal of Hematotherapy & Stem Cell Research", volume = "12", number = "6", pages = "689-699", month = "December", year = "2003", doi = "10.1089/15258160360732713", issn = "1525-8165", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150202-100311553", note = "© Mary Ann Liebert, Inc. 2003\n\nReceived September 30,2003; accepted October 24, 2003.\n\nThe authors are generously supported by the Swedish Research Council, the Norwegian Foundation for Health and Rehabilitation, National Institutes of Health, National Science Foundation, CNS Margot Anderson Foundation, and the Baxter Family Foundation.", revision_no = "14", abstract = "Cellular transplantation therapy is thought to play a central role in the concept of restorative neurosurgery, which aims to restore function to the damaged nervous system. Stem cells represent a potentially renewable source of transplantable cells. However, control of the behavior of these cells, both in the process of clonogenic expansion and post-transplantation, represents formidable challenges. Stem cell behavior is thought to be directed by extracellular signals in their in vivo niches, many of which are protein or peptide based. As only one example, activation of Notch plays an important role in normal development and is the strongest known signal for stem cells to choose glial over neuronal fates. Therefore, artificial extracellular matrix proteins represent a potentially powerful tool to custom design artificial niches to strategically control stem cell behavior. We have developed a family of aECM proteins that incorporate the active domains of the DSL ligands to the Notch receptor into an elastin-based backbone. The development of our DSL-elastin artificial proteins demonstrates the design strategy and methodology for the production of bioactive artificial extracellular matrix proteins aimed at modulating\nstem cell behavior, and this method can be used to design other bioactive aECM proteins. In addition, we have developed a method for the isolation and characterization of adult human neural stem cells from periventricular tissue harvested from living patients. This paper reviews cellular transplantation therapy from the clinical perspective and summarizes ongoing work aimed at exploring the intriguing possibility of autologous transplantation, whereby neural stem cells can be harvested from adult patients, expanded or modified in vitro in artificial niches, and retransplanted into the original patient.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53311, title ="Non-canonical amino acids in protein engineering", author = "Link, A. James and Mock, Marissa L.", journal = "Current Opinion in Biotechnology", volume = "14", number = "6", pages = "603-609", month = "December", year = "2003", doi = "10.1016/j.copbio.2003.10.011", issn = "0958-1669", url = "https://resolver.caltech.edu/CaltechAUTHORS:LINcob2003", note = "© 2003 Elsevier. \n\nWork on non-canonical amino acids at Caltech has been supported by grants from the National Science Foundation, the National Institutes of Health and the US Army Research Office.", revision_no = "10", abstract = "Methods for engineering proteins that contain non-canonical amino acids have advanced rapidly in the past few years. Novel amino acids can be introduced into recombinant proteins in either a residue-specific or site-specific fashion. The methods are complementary: residue-specific incorporation allows engineering of the overall physical and chemical behavior of proteins and protein-like macromolecules, whereas site-specific methods allow mechanistic questions to be probed in atomistic detail. Challenges remain in the engineering of the translational apparatus and in the design of schemes that can be used to encode both canonical and non-canonical amino acids.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53477, title ="Mechanism for the Phase Transition of a Genetically Engineered Elastin Model Peptide (VPGIG)_(40) in Aqueous Solution", author = "Yamaoka, Tetsuji and Tamura, Takumi", journal = "Biomacromolecules", volume = "4", number = "6", pages = "1680-1685", month = "November", year = "2003", doi = "10.1021/bm034120l", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150109-113118565", note = "© 2003 American Chemical Society. \n\nPublished In Issue November 10, 2003. Publication Date (Web): September 5, 2003. Received April 19, 2003. Revised Manuscript Received August 5, 2003.", revision_no = "14", abstract = "The concentration dependence of the pressure- and temperature-induced cloud point transition (P_c and T_c, respectively) of aqueous solutions of an elastin-like polypeptide with a repeating pentapeptide Val−Pro−Gly−Ile−Gly sequence (MGLDGSMG(VPGIG)_(40)VPLE) was investigated by using apparent light scattering, differential scanning calorimetry, and circular dichroism methods. In addition, the effects of salts and surfactants on these properties were investigated. The P_c and T_c of the present peptide in aqueous solution were strongly concentration dependent. The calorimetric measurements showed that the enthalpy of transitions was 300−400 kJ/mol, i.e., 7−10 kJ/mol per VPGIG pentamer. The T_c of the (VPGIG)_(40) solution was highly affected by the addition of inert salts or SDS. The effects of salts were consistent with those observed in the lyotropic series or Hoffmeister series. The CD spectrum at low peptide concentrations indicated that the present peptide forms type II β-turn-like structure(s) at higher temperatures, but the temperature dependence of random coil diminishment (195 nm) and β-turn formation (210 nm) were not exactly coincident. A hypothetical mechanism of the (VPGIG)_(40) phase transition that could account for these observations was postulated. Observations suggest that the temperature-responsive properties of the elastin model peptides occur via a mechanism involving conformational change−association−aggregation and that the first two are strongly interactive. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/54039, title ="Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins", author = "Heilshorn, Sarah C. and DiZio, Kathleen A.", journal = "Biomaterials", volume = "24", number = "23", pages = "4245-4252", month = "October", year = "2003", doi = "10.1016/S0142-9612(03)00294-1", issn = "0142-9612", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150123-160702434", note = "© 2003 Elsevier Ltd.\nReceived 16 December 2002; accepted 8 April 2003.\n\nAcknowledgements: We thank Paul Nowatzki for expression and purification of aECM 3, Krystle Wang and Summer Zhang for help with the lipopolysaccharide assays, Sarah Wilhoit and Michelle Giron for help with the competitive inhibition binding assays, and Julie Liu for helpful discussions. Funding was provided by the National Institutes of Health and the National Science Foundation.", revision_no = "10", abstract = "This study examines the spreading and adhesion of human umbilical vein endothelial cells (HUVEC) on artificial extracellular matrix (aECM) proteins containing sequences derived from elastin and fibronectin. Three aECM variants were studied: aECM 1 contains lysine residues periodically spaced within the protein sequence and three repeats of the CS5 domain of fibronectin, aECM 2 contains periodically spaced lysines and three repeats of a scrambled CS5 sequence, and aECM 3 contains lysines at the protein termini and five CS5 repeats. Comparative cell binding and peptide inhibition assays confirm that the tetrapeptide sequence REDV is responsible for HUVEC adhesion to aECM proteins that contain the CS5 domain. Furthermore, more than 60% of adherent HUVEC were retained on aECM 1 after exposure to physiologically relevant shear stresses (⩽100 dynes/cm^2). Finally, the levels of thrombogenic markers (tissue plasminogen activator and plasminogen activator inhibitor–1) secreted by HUVEC monolayers on aECM 1 were found to be similar to those secreted by HUVEC monolayers cultured on fibronectin. These characteristics, along with the physical strength and elasticity of crosslinked films prepared from these materials, make aECM proteins promising candidates for application in small-diameter vascular grafts.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53312, title ="Internal segregation and side chain ordering in hairy-rod polypeptide monolayers at the gas/water interface: An x-ray scattering study", author = "Fukuto, Masafumi and Heilmann, Ralf K.", journal = "Journal of Chemical Physics", volume = "119", number = "12", pages = "6253", month = "September", year = "2003", doi = "10.1063/1.1602058", issn = "0021-9606", url = "https://resolver.caltech.edu/CaltechAUTHORS:FUKjcp2003", note = "© 2003 American Institute of Physics. \n\n(Received 19 May 2003; accepted 27 June 2003) \n\nThe Harvard contribution to this work was supported by Grant No. NSF-DMR-01-24936. D.T. acknowledges support by Grant No. NSF-DMR-01-10437. NSLS at Brookhaven National Laboratory is supported by Grant No. DE-AC02-76CH00016.", revision_no = "9", abstract = "We report studies of the structure and packing of Langmuirmonolayers (LMs) of polypeptide poly(γ-4-(n-hexadecyloxy)benzyl α,L-glutamate) (C16–O–PBLG) on the surface of water. The molecule is a “hairy rod” and consists of side attachments of hexadecyloxy chains (–O–C16) to the rigid rod-like core made up of α-helical poly(γ-benzyl L-glutamate) (PBLG). Measurements include surface pressure (Π) versus area/monomer (A) isotherms, x-ray specular reflectivity (XR), and grazing incidence diffraction(GID). In contrast to the LM of bare PBLG on water, which undergoes a monolayer/bilayer transition with increasing Π, monolayers of C16–O–PBLG remain stable up to the highest densities. On the basis of XR and GID results, the structure of the C16–O–PBLG monolayer is characterized by the following main features. First, hydrophobicity causes the –O–C16 chains to segregate towards the film/gas interface and away from water and the PBLG cores, which sit parallel to and near the water/film interface. Since the attachment position of some of the side chains is at the core/water interface, the segregation forces these chains into the space between neighboring core rods. Compression associated with increasing Π thickens the film but the internally segregated structure is maintained for all Π (i.e., >∼30 dyne/cm). Second, the C16–O–PBLG rods form domains in which the rods are aligned parallel to each other and to the interface. The correlation length for the interhelix positional order of the rods is short and typically comparable to or less than the length of the rods. With increasing Π the spacing d between nearest-neighbor rods decreases linearly with A at high Π, indicating a direct correspondence between the macroscopic compressibility and the microscopic interhelix compressibility. Third, as Π increases past ∼5 dyne/cm, the local packing of tethered –O–C16 chains displays the same herringbone (HB) order that is common for high-density bulk and monolayer phases of alkyl chains. Various features of the observed GID peaks also imply that the HB order of –O–C16 chains is oriented with respect to the helical axes of aligned PBLG cores. We propose that the HB order is established initially by one-dimensionally confined chains between aligned rods at low Π and grows laterally with compression.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53314, title ="Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity", author = "Cirino, Patrick C. and Tang, Yi", journal = "Biotechnology and Bioengineering", volume = "83", number = "6", pages = "729-734", month = "September", year = "2003", doi = "10.1002/bit.10718", issn = "0006-3592", url = "https://resolver.caltech.edu/CaltechAUTHORS:CIRbb2003", note = "© 2003 Wiley. \n\nReceived 17 December 2002; accepted 24 March 2003. Published online 23 June 2003. \n\nThe authors thank Dr. Mona Shahgholi for assistance with the\nMALDI-TOF mass spectral analyses and the Yale E. coli Genetic\nStock Center for providing the methionine auxotroph.\n\nContract grant sponsors: Biotechnology Research and Development\nCorporation (Peoria, IL); NSF Graduate Research Fellowship; NIH Contract grant number: R01-GM62523-02", revision_no = "10", abstract = "In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53823, title ="Cell Surface Labeling of Escherichia coli via Copper(I)-Catalyzed [3+2] Cycloaddition", author = "Link, A. James and Tirrell, David A.", journal = "Journal of the American Chemical Society", volume = "125", number = "37", pages = "11164-11165", month = "September", year = "2003", doi = "10.1021/ja036765z", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-104839242", note = "Copyright © 2003 American Chemical Society.\nPublished In Issue September 17, 2003. Publication Date (Web): August 23, 2003 Received June 18, 2003.\n\nAcknowledgment. We thank K. Barry Sharpless and Valery\nFokin for their gift of the triazole ligand and for helpful advice on\nthe azide-alkyne cycloaddition. We also thank Isaac Carrico, Pin\nWang, and Rochelle Diamond for helpful discussions and Rich\nRoberts for use of his flow cytometer. This work was supported\nby the NSF Center for the Science and Engineering of Materials at\nCaltech. A.J.L. is an NSF Graduate Research Fellow.\n\nSupporting Information Available: Experimental protocols and\ndetails about flow cytometry (PDF).", revision_no = "14", abstract = "Labeling of the cell surface of Escherichia coli was accomplished by expression of a recombinant outer membrane protein, OmpC, in the presence of the unnatural amino acid azidohomoalanine, which acts as a methionine surrogate. The surface-exposed azide moieties of whole cells were biotinylated via Cu(1)-catalyzed [3+2] azide-alkyne cycloaddition. The specificity of labeling of both wild-type OmpC and a mutant containing additional methionine sites for azidohomoalanine incorporation was confirmed by Western blotting. Flow cytometry was performed to examine the specificity of the labeling. Cells that express the mutant form of OmpC in the presence of azidohomoalanine, which were biotinylated and stained with fluorescent avidin, exhibit a mean fluorescence 10-fold higher than the background. Incorporation of an unnatural amino acid can thus be determined on a single-cell basis. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53818, title ="Breaking the degeneracy of the genetic code", author = "Kwon, Inchan and Kirshenbaum, Kent", journal = "Journal of the American Chemical Society", volume = "125", number = "25", pages = "7512-7513", month = "June", year = "2003", doi = "10.1021/ja0350076 ", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-102631008", note = "Copyright © 2003 American Chemical Society.\nPublished In Issue June 25, 2003. Publication Date (Web): May 29, 2003. Received March 5, 2003.\n\nAcknowledgment. This work was supported by NIH GM62523\nand by the NSF MRSEC program. We thank Dr. Mona Shahgholi,\nDr. Gary M. Hathaway, and Dr. Jie Zhou for mass spectrometry\nstudies. We thank Pin Wang and Soojin Son for helpful discussions.\n\nSupporting Information Available: Protein expression gels and\nLC/MS/MS spectra (PDF).\n\nAlso found in: Conference: 225th National Meeting of the American-Chemical-Society Location: New Orleans, LA. Date: MAR 23-27, 2003. Sponsor(s): Amer Chem Soc. Abstracts of Papers of the American Chemical Society. Volume: 225. Pages: U322-U322. Part: 2. Meeting Abstract: 255-ORGN. Published: MAR 2003. ", revision_no = "12", abstract = "A mutant yeast phenylalanine transfer RNA (ytRNA^(Phe)_(AAA)) containing a modified (AAA) anticodon was generated to explore the feasibility of breaking the degeneracy of the genetic code in Escherichia coli. By using an E. coli strain co-transformed with ytRNA^(Phe)_(AAA) and a mutant yeast phenylalanyl-tRNA synthetase, we demonstrate efficient replacement of phenylalanine (Phe) by L-3-(2-naphthyl)alanine (Nal) at UUU, but not at UUC codons. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53819, title ="Incorporation of Trifluoroisoleucine into Proteins in Vivo", author = "Wang, Pin and Tang, Yi", journal = "Journal of the American Chemical Society", volume = "125", number = "23", pages = "6900-6906", month = "June", year = "2003", doi = "10.1021/ja0298287", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-102631096", note = "Copyright © 2003 American Chemical Society.\nPublished In Issue June 11, 2003. Publication Date (Web): May 17, 2003. Received December 19, 2002.\n\nAcknowledgment. This work was supported by NIH grant\nR01-GM62523, and by the NSF Center for the Science and\nEngineering of Materials at Caltech. We are grateful to Lili Yang from David Baltimore’s laboratory at Caltech and Dr. Susan Kovats at City of Hope for assistance on the cell-proliferation assay. We thank Dr. Osamu Nureki at the University of Tokyo for providing coordinates of the crystal structure of IleRS complexed with isoleucine, and Kent Kirshenbaum for helpful discussions.", revision_no = "7", abstract = "Two fluorinated derivatives of isoleucine:\u2009 d,l-2-amino-3-trifluoromethyl pentanoic acid (3TFI, 2) and d,l-2-amino-5,5,5-trifluoro-3-methyl pentanoic acid (5TFI, 3) were prepared. 5TFI was incorporated into a model target protein, murine dihydrofolate reductase (mDHFR), in an isoleucine auxotrophic Escherichia coli host strain suspended in 5TFI-supplemented minimal medium depleted of isoleucine. Incorporation of 5TFI was confirmed by tryptic peptide analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) of the protein product. Amino acid analysis showed that more than 93% of the encoded isoleucine residues were replaced by 5TFI. Measurement of the rate of activation of 5TFI by the E. coli isoleucyl-tRNA synthetase (IleRS) yielded a specificity constant (k_(cat)/K_m) 134-fold lower than that for isoleucine. 5TFI was successfully introduced into the cytokine murine interleukin-2 (mIL-2) at the encoded isoleucine positions. The concentration of fluorinated protein that elicits 50% of the maximal proliferative response is 3.87 ng/mL, about 30% higher than that of wild-type mIL-2 (EC_(50) = 2.70 ng/mL). The maximal responses are equivalent for the fluorinated and wild-type cytokines, indicating that fluorinated proteins can fold into stable and functional structures. 3TFI yielded no evidence for in vivo incorporation into recombinant proteins, and no evidence for activation by IleRS in vitro. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53749, title ="Engineering of the Extracellular Matrix: Working toward Neural Stem Cell Programming and Neurorestoration — Concept and Progress Report", author = "Liu, Charles Y. and Apuzzo, Michael L. J.", journal = "Neurosurgery", volume = "52", number = "5", pages = "1154-1167", month = "May", year = "2003", doi = "10.1227/01.NEU.0000057747.93237.8F", issn = "0148-396X", url = "https://resolver.caltech.edu/CaltechAUTHORS:LIUn2003", note = "© 2003 the Congress of Neurological Surgeons. \n\nReceived November 6, 2002. Accepted January 8, 2003. \n\nThis work was generously supported by Grant R01-HL59987-03 from the National Institutes of Health, Grant BES-9901648 from the National Science Foundation, a gift from Baxter Healthcare, and the CNS Margot Anderson Foundation. We do not have any financial interest in the subject matter discussed.", revision_no = "11", abstract = "In the concept of neurorestoration, cellular and structural elements that have been lost are replaced, and their function is restored. Central to this therapeutic strategy is the transplantation of neural progenitor cells such as clonogenically expanded stem cells. Stem cells make decisions regarding fate and patterning in response to external environmental signals. The therapeutic effectiveness of neural stem cells may be facilitated by the ability to manipulate these signals in a temporal and spatially appropriate fashion. Artificial deoxyribonucleic acid and artificial protein technology combines elements of protein engineering, molecular biology, and recombinant deoxyribonucleic acid technology to produce proteins with functional domains derived from naturally occurring proteins and represents a potentially powerful tool to modulate stem cell behavior. To this end, we have developed three artificial extracellular matrix proteins that incorporate the active domain of hJagged1 and hDelta1 into an elastin backbone. hJagged1 and hDelta1 are members of the DSL family of ligands to the Notch receptor, a signaling system that is very important in development and is the strongest known signal to instruct neural progenitor cells to choose glial fates over neuronal fates. The successful cloning of the artificial genes was confirmed by test digestions with appropriate restriction enzymes as well as direct deoxyribonucleic acid sequencing. In addition, we have demonstrated that all three artificial extracellular matrix proteins express at a high level in a prokaryotic host. This report describes the concept and progress in an entirely novel and previously unreported approach to modulate neural stem cell behavior. Its future application could include in vitro processing of stem cells before transplantation, supporting and programming the cells after transplantation, as well as the development of bioactive biomaterials.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53341, title ="Mechanical Properties of Artificial Protein Matrices Engineered for Control of Cell and Tissue Behavior", author = "Di Zio, Kathleen and Tirrell, David A.", journal = "Macromolecules", volume = "36", number = "5", pages = "1553-1558", month = "March", year = "2003", doi = "10.1021/ma0256587", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:ZIOm2003", note = "Copyright © 2003 American Chemical Society. \n\nReceived September 9, 2002; Revised Manuscript Received January 2, 2003. Publication Date (Web): February 14, 2003. \n\nThis work was supported by NIH Grant 5 RO1 HL59987-03 and by NSF Grant BES-9901648. Mass spectra were recorded in the central facility for mass spectrometry of the Center for the Science and Engineering of Materials at Caltech.", revision_no = "10", abstract = "Genetic engineering methods were used for the preparation of artificial proteins containing sequences designed to reproduce essential features of the extracellular matrix (ECM). The long-term objective of the work is to develop matrices for use in the engineering of small-diameter vascular grafts. The CS5 domain of fibronectin provides binding sites for vascular endothelial cells, while an elastin-like repeat, [(VPGIG)_2(VPGKG)(VPGIG)_2], controls the mechanical properties and includes sites for covalent cross-linking. Bis(sulfosuccinimidyl) suberate and disuccinimidyl suberate were used to cross-link artificial ECM protein films for uniaxial tensile testing. Variation in the amount of cross-linker and protein weight fraction allowed preparation of films with Young's moduli ranging from 0.07 to 0.97 MPa. The weight fraction of protein in the hydrated, cross-linked films was measured to be between 0.2 and 0.4; the molecular weight between cross-links (M_c) varied from 3000 to 38\u2009000. The moduli and M_c of the films span the ranges reported for natural elastins.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53845, title ="Virtual Screening for Binding of Phenylalanine Analogues to Phenylalanyl-tRNA Synthetase", author = "Wang, Pin and Vaidehi, Nagarajan", journal = "Journal of the American Chemical Society", volume = "124", number = "48", pages = "14442-14449", month = "December", year = "2002", doi = "10.1021/ja0175441", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-152910657", note = "Copyright © 2002 American Chemical Society.\nPublished In Issue December 04, 2002. Publication Date (Web): November 12, 2002. Received November 14, 2001. Revised July 30, 2002.\n\nAcknowledgment. This work was supported by NIH/BRG\nR01-GM62523 (D.A.T., W.A.G., and N.V.) and by the Center\nfor the Science and Engineering of Materials at Caltech (NSF-MRSEC).\nThe facilities of the Materials and Process Simulation\nCenter used in this project are supported also by DOE (ASCI\nASAP), NSF (CTS and MRI), NIH, ARO-MURI, Chevron\nCorp., MMM, Seiko-Epson, Dow Chemical, Avery-Dennison\nCorp., Kellogg’s, General Motors, Asahi Kasei, the Beckman\nInstitute, and ONR.", revision_no = "9", abstract = "Although incorporation of nonnatural amino acids provides a powerful means of controlling protein structure and function, experimental investigations of amino acid analogues for utilization by the protein biosynthetic machinery can be costly and time-consuming. In this paper, we describe a computational protocol (HierDock) for predicting the relative energies of binding of phenylalanine analogues to phenylalanyl-tRNA synthetase (PheRS). Starting with the crystal structure of Thermus thermophilus PheRS without bound ligand, HierDock predicts the binding site of phenylalanine (Phe) within 1.1 Å of that revealed by the crystal structure of PheRS cocrystallized with Phe. The calculated binding energies of Phe analogues in PheRS, using HierDock, correlate well with the translational activities of the same analogues in Escherichia coli. HierDock identifies p-fluorophenylalanine and 3-thienylalanine as especially good substrates for PheRS, in agreement with experiment. These results suggest that the HierDock protocol may be useful for virtual screening of amino acid analogues prior to experiment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53349, title ="Attenuation of the Editing Activity of the Escherichia coli Leucyl-tRNA Synthetase Allows Incorporation of Novel Amino Acids into Proteins in Vivo", author = "Tang, Yi and Tirrell, David A.", journal = "Biochemistry", volume = "41", number = "34", pages = "10635-10645", month = "August", year = "2002", doi = "10.1021/bi026130x", issn = "0006-2960", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150108-164212113", note = "© 2002 American Chemical Society\n\nThis work was supported by the NSF Center for the Science and Engineering of Materials at Caltech and by NIH Grant R01-GM62523. Y.T. is supported by a graduate fellowship from the Whitaker Foundation.\n\nWe thank Dr. Mona Shahgholi and Dr. Gary Hathaway for assistance with typtic digest mass spectrometry. We are grateful to Dr. Anthony Bishop from Prof. Schimmel’s group for his advice on aminoacylation assays. We also thank Prof. Stephen Cusack for providing coordinates of the T. thermophilus LeuRS crystal structure coordinates and P. Wang, I. Carrico, and K. Kirshenbaum for fruitful discussions.", revision_no = "10", abstract = "The fidelity of translation is dependent on the specificity of the aminoacyl-tRNA synthetases (aaRSs). The aaRSs that activate the hydrophobic amino acids leucine, isoleucine, and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs. Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis on the basis of the size and hydrophilicity of the amino acid side chain. Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA^(Leu) by isoleucine and methionine. Thr252 of Escherichia coli LeuRS has been shown previously to be important in defining the size of the editing cavity. Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F). The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced. The rate of misaminoacylation of tRNA^(Leu) by isoleucine and valine increases with the increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at the leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E. coli. In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine, and 2-butynylalanine all support protein synthesis in E. coli hosts harboring the mutant synthetase. These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53847, title ="Assembly of Triple-Stranded β-Sheet Peptides at Interfaces", author = "Rapaport, Hanna and Möller, Gunter", journal = "Journal of the American Chemical Society", volume = "124", number = "32", pages = "9342-9343", month = "August", year = "2002", doi = "10.1021/ja026765j", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-153735804", note = "Copyright © 2002 American Chemical Society. \nPublished In Issue August 14, 2002. Publication Date (Web): July 19, 2002. Received May 2, 2002.\n\nWe thank Suzanna Horvath and her staff\nfor peptide synthesis, George Rossman for help in ATR-FTIR, and\nHASYLAB for synchrotron beamtime. This work was supported\nby the U.S.-Israel Binational Science Foundation, the U.S. National\nScience Foundation, the DanSync program of the Danish Natural\nScience Research Council, and the European Community, TMR Contract\nERBFMGECT950059.\n\nSupporting Information Available: Surface pressure-area isotherms,\ndescription of GIXD measurements, FTIR spectra, additional\nBAM images of BS30 (PDF).", revision_no = "14", abstract = "A 30-residue peptide, BS30, which incorporates two proline residues to induce reverse turns, was designed to form a triple-stranded β-sheet monolayer at the air−water interface. To discern the structural role of proline, a second peptide, BS30G, identical to BS30 but with glycine residues replacing proline, was prepared and examined in parallel fashion. Surface pressure−molecular area isotherms indicated a limiting area per molecule (ca. 460 Å^2) for BS30 that corresponds well to that estimated from the known dimensions of crystalline β-sheet monolayers (492 Å^2). Comparable measurements on BS30G yielded a smaller molecular area (380 Å^2). Grazing incidence X-ray diffraction measurements performed on the BS30 monolayer at nominal area per molecule of 500 Å^2, exhibited two Bragg peaks corresponding to 4.79 and 34.9 Å spacings, consistent with formation of triple-stranded β-sheet structures that assemble into two-dimensional crystallites at the air−water interface. Visualized by Brewster angle microscopy, BS30 monolayers displayed uniform, solidlike domains, whereas BS30G appeared to be disordered.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53347, title ="Confinement-induced order of tethered alkyl chains at the water/vapor interface", author = "Fukuto, M. and Heilmann, R. K.", journal = "Physical Review E", volume = "66", number = "1", pages = "Art. No. 010601~R!", month = "July", year = "2002", doi = "10.1103/PhysRevE.66.010601", issn = "1539-3755", url = "https://resolver.caltech.edu/CaltechAUTHORS:FUKpre2003", note = "© 2003 American Physical Society. \n\n(Received 9 November 2001; published 23 July 2002) \n\nThe Harvard contribution to this work was supported by Grant No. NSF-DMR-01-24936. D.T. acknowledges support by Grant No. NSF-DMR-01-10437. NSLS at Brookhaven National Laboratory is supported by No. DE-AC02-76CH00016.", revision_no = "8", abstract = "Packing of tethered alkyl chains in Langmuir monolayers of a hairy-rod polypeptide poly[γ-4-(n-hexadecyloxy)benzyl α,L-glutamate] on water has been studied by x-ray scattering measurements at room temperature. The rods lie parallel to the surface while the alkyl side chains segregate toward the vapor. Results indicate that the herringbone order of the alkyl chains is established initially by one-dimensionally confined chains between aligned rods and grows laterally with compression.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/24112, title ="A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins", author = "Datta, Deepshikha and Wang, Pin", journal = "Journal of the American Chemical Society", volume = "124", number = "20", pages = "5652-5653", month = "May", year = "2002", doi = "10.1021/ja0177096 ", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20110620-160431532", note = "© 2002 American Chemical Society.\n\nReceived December 7, 2001.\nPublication Date (Web): April 26, 2002.\n\nThe authors thank William A. Goddard III,\nNagarajan Vaidehi, Kent Kirshenbaum, and Yi Tang for helpful\ndiscussions. This work was supported by NIH Grants R01-GM62523 and T32-GM08501, the NSF Center for the Science and\nEngineering of Materials at Caltech, the Howard Hughes Medical\nInstitute, the Ralph M. Parsons Foundation, and an IBM shared\nUniversity Research Grant.", revision_no = "20", abstract = "Incorporation of non-natural amino acids into proteins in vivo expands the scope of protein synthesis and design. p-Acetylphenylalanine was incorporated into recombinant dihydrofolate reductase (DHFR) in Escherichia coli via a computationally designed mutant form of the phenylalanyl-tRNA synthetase of the host. DHFR outfitted with ketone functionality can be chemoselectively ligated with hydrazide reagents under mild conditions.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/69354, title ="Incorporation of unsaturated isoleucine analogues into proteins in vivo", author = "Michon, Thierry and Barbot, Francis", number = "276", pages = "63-72", month = "April", year = "2002", doi = "10.1039/9781847551672-00063", issn = "2042-0625", isbn = "978-0-85404-856-4", url = "https://resolver.caltech.edu/CaltechAUTHORS:20160801-122125209", note = "© 2002 Royal Society of Chemistry. \n\nThe authors thank NATO for its financial support to T. M.'s year-long sabbatical. We are grateful to Kristi Kiick for fruitful discussions.", revision_no = "11", abstract = "The translational activity of various unsaturated analogs of L-isoleucine was evaluated using an Escherichia coli strain auxotrophic for isoleucine. It was observed that the alkene [2-amino-3-methyl-4-pentenoic acid (2)] and alkyne [2-amino-3-rnethyl-4-pentynoic acid (3)] derivatives of L-isoleucine can support protein synthesis at levels approximately 50% of that observed in cultures supplemented with isoleucine. However, no incorporation of the αC or βC methylated derivatives could be detected. In order to examine the stereoselectivity of incorporation, the (2S, 3s) and (2S, 3R) diastereomers of 2 and 3 were prepared. The extents of isoleucine substitution in vivo were 80% and 70% for (2S, 3S)-2 and (2S, 3S)-3, respectively, under the conditions examined in this study. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/830, title ="Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation", author = "Kiick, Kristi L. and Saxon, Eliana", journal = "Proceedings of the National Academy of Sciences of the United States of America", volume = "99", number = "1", pages = "19-24", month = "January", year = "2002", doi = "10.1073/pnas.012583299", issn = "0027-8424", url = "https://resolver.caltech.edu/CaltechAUTHORS:KIIpnas02", note = "© 2002 by the National Academy of Sciences \n\nCommunicated by Ralph F. Hirschmann, University of Pennsylvania, Philadelphia, PA, October 31, 2001 (received for review August 15, 2001). Published online before print December 18, 2001, 10.1073/pnas.012583299 \n\nWe acknowledge the generous donation of plasmids encoding MetRS from H. Jakubowski and Y. Mechulam and the assistance of J. Kua in modeling the azide-functionalized amino acids. This research was supported by the Office of Naval Research, Grant N00014–98-1–0605 and Order N00014–98-F-0402 through the U.S. Department of Energy under Contract DE-AC03–76SF00098, the National Institutes of Health (GM58867–01), the Polymers and Genetics Programs of the U.S. National Science Foundation, and the U.S. Army Research Office. K.L.K. thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship. E.S. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. The Center for New Directions in Organic Synthesis is supported by Bristol-Myers Squibb as a supporting member. \n\nK.L.K. and E.S. contributed equally to this work. \n\nThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.", revision_no = "10", abstract = "The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/27632, title ="Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences \n", author = "Mawn, Mary V. and Fournier, Maurille J.", journal = "Journal of Bacteriology", volume = "184", number = "2", pages = "494-502", month = "January", year = "2002", doi = "10.1128/JB.184.2.494-502.2002 ", issn = "0021-9193", url = "https://resolver.caltech.edu/CaltechAUTHORS:20111104-135322237", note = "© 2002 American Society for Microbiology. \n\nReceived 31 July 2001; Accepted 16 October 2001. \n\nThis work was supported by grants from the NSF Polymers and Genetics Programs and by the NSF Materials Research Science and Engineering Center at the University of Massachusetts. We thank Vincent Conticello for early contributions to this work and Paul Bode for his mathematical analysis of sucrose gradient profiles.", revision_no = "16", abstract = "We have constructed synthetic coding sequences for the expression of poly(α,l-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5′-GAGGAGG-3′) that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53682, title ="Biosynthesis of Proteins Incorporating a Versatile Set of Phenylalanine Analogues", author = "Kirshenbaum, Kent and Carrico, Isaac S.", journal = "ChemBioChem", volume = "3", number = "2-3", pages = "235-237", month = "January", year = "2002", doi = "10.1002/1439-7633(20020301)3:2/3<235::AID-CBIC235>3.0.CO;2-7", issn = "1439-4227", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-094948843", note = "© 1999-2014 John Wiley & Sons, Inc. \nIssue published online: 7 MAR 2002.\nArticle first published online: 7 MAR 2002.\nManuscript Received: 10 OCT 2001.\n\nThis work was supported by the National Institutes of Health through a grant (no.: RO1-GM62523-01), a postdoctoral fellowship to K.K. (no.: F32-GM29474), and a training grant stipend to I.S.C. (no.: T32-GM08501). We thank Nandita Sharma, Ian Suydam, Steven Boxer, Melanie Bennett, Mona Shahgholi, Hanna Rapaport, and Yi Tang for their advice and assistance.", revision_no = "9", abstract = "Unnatural amino acids with useful chemical functionality can replace phenylalanine in bacterial proteins. Coexpression of a promiscuous phenylalanine-tRNA synthetase mutant enables the synthesis of target proteins bearing iodophenyl, cyanophenyl, ethynylphenyl, azidophenyl, and pyridyl groups (see general structures). Proteins incorporating the analogues have a range of potential applications, including Pd-mediated conjugation (R=CCH), photoaffinity labeling (R=N_3), X-ray phasing (R=I), and novel metal coordination (R=pyridyl).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53355, title ="Dynamic Structure of a Protein Hydrogel: A Solid-State NMR Study", author = "Kennedy, S. B. and deAzevedo, E. R.", journal = "Macromolecules", volume = "34", number = "25", pages = "8675-8685", month = "December", year = "2001", doi = "10.1021/ma010768j", issn = "0024-9297", url = "https://resolver.caltech.edu/CaltechAUTHORS:KENm2001", note = "Copyright © 2001 American Chemical Society. \n\nReceived May 4, 2001. Publication Date (Web): November 7, 2001. \n\nThis work was partially supported by the NSF Materials Science and Engineering Center at the University of Massachusetts, Amherst. M. Hong acknowledges NSF for a POWRE award and the Beckman Foundation for a Young Investigator Award. E. R. deAzevedo thanks the FAPESP-Brazil program for a graduate fellowship.", revision_no = "8", abstract = "^(13)C and ^(15)N solid-state NMR spectroscopy has been used to study the dynamic structure of a genetically engineered multidomain protein hydrogel that contains two leucine-zipper domains and a central polyelectrolyte domain. ^(13)C NMR spectra show that on the microsecond time scale the central domain is isotropically mobile while the leucine-zipper domains are rigid. This supports the hypothesis that the central domain acts as the flexible swelling agent of the gel network while the terminal domains form intermolecular aggregates. ^(13)C isotropic chemical shifts indicate that the terminal domains are helical, while the central domain has a random coil conformation. On the millisecond time scale, the leucine-zipper domains are highly dynamic, as determined from the ^(13)C-detected ^(15)N CODEX experiment. The motion is rigid-body in nature with a correlation time of about 80 ms at room temperature and has an average amplitude of about 50°. Several specific motional models are considered by comparing simulated and experimental exchange intensities as a function of the recoupling time for ^(15)N chemical shift anisotropy. The experimental data are consistent with two of the models considered:\u2009 a random jump model and a uniaxial rotation model. The implications of this motion to strand exchange between helical bundles are discussed.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53848, title ="Biosynthesis of a Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine in an Engineered Bacterial Host", author = "Tang, Yi and Tirrell, David A.", journal = "Journal of the American Chemical Society", volume = "123", number = "44", pages = "11089-11090", month = "November", year = "2001", doi = "10.1021/ja016652k", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-154847107", note = "Copyright © 2001 American Chemical Society.\nPublished In Issue November 07, 2001. Publication Date (Web): October 11, 2001. Received July 18, 2001.\n\nAcknowledgment. This work was supported by the NSF MRSEC\nprogram at Caltech. Y.T. is supported by a fellowship from the Whitaker\nFoundation. We thank Dr. Giovanna Ghirlanda and Prof. William\nDeGrado for ultracentrifugation studies. We also thank Kent Kirshenbaum,\nIsaac Carrico, and Pin Wang for insightful discussions.\n\nSupporting Information Available: Amino acid synthesis, plasmid\nconstruction, protein expression, and protein analysis (PDF).", revision_no = "12", abstract = "Incorporation of nonnatural amino acid residues allows engineering\nof proteins with novel chemical functionality and unusual\nphysical properties. We have shown recently that coiled-coil\nproteins prepared in vivo can be stabilized significantly by\nreplacement of leucine by trifluoroleucine (1). In the same series\nof experiments, however, we were unsuccessful in our attempts\nto incorporate the more highly fluorinated analogue hexafluoroleucine\n(2). We report here that modification of the leucyl-tRNA\nsynthetase (LeuRS) activity of the host allows efficient incorporation\nof 2 into recombinant proteins prepared in Escherichia coli.\nFurthermore, the coiled-coil protein used to demonstrate incorporation\nof 2 exhibits enhanced stability in comparison to the\nsame protein enriched in 1, possibly due to the increased\nhydrophobic character of the additional trifluoromethyl group in\nthe protein core.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/27623, title ="Protein-based materials, toward a new level of structural control", author = "van Hest, Jan C. M. and Tirrell, David A.", journal = "Chemical Communications", volume = "2001", number = "19", pages = "1897-1904", month = "October", year = "2001", doi = "10.1039/b105185g ", issn = "1359-7345", url = "https://resolver.caltech.edu/CaltechAUTHORS:20111104-083950931", note = "© 2001 The Royal Society of Chemistry. Received (in Cambridge, UK) 13th June 2001, Accepted 2nd August 2001.\nFirst published as an Advance Article on the web 18th September 2001. The authors would like to thank Jurry Hannink for his assistance with the schematic representations and front cover design.", revision_no = "13", abstract = "Through billions of years of evolution nature has created and refined structural proteins for a wide variety of specific purposes. Amino acid sequences and their associated folding patterns combine to create elastic, rigid or tough materials. In many respects, nature’s intricately designed products provide challenging examples for materials scientists, but translation of natural structural concepts into bio-inspired materials requires a level of control of macromolecular architecture far higher than that afforded by conventional polymerization processes. An increasingly important approach to this problem has been to use biological systems for production of materials. Through protein engineering, artificial genes can be developed that encode protein-based materials with desired features. Structural elements found in nature, such as β-sheets and α-helices, can be combined with great flexibility, and can be outfitted with functional elements such as cell binding sites or enzymatic domains. The possibility of incorporating non-natural amino acids increases the versatility of protein engineering still further. It is expected that such methods will have large impact in the field of materials science, and especially in biomedical materials science, in the future.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53356, title ="Self-Association and Membrane-Binding Behavior of Melittins Containing Trifluoroleucine", author = "Niemz, Angelika and Tirrell, David A.", journal = "Journal of the American Chemical Society", volume = "123", number = "30", pages = "7407-7413", month = "August", year = "2001", doi = "10.1021/ja004351p", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:NIEjacs2001", note = "Copyright © 2001 American Chemical Society. \n\nReceived December 27, 2000. Publication Date (Web): July 10, 2001. \n\nWe would like to acknowledge Dr. Suzanna Horvath, Director of the Caltech Biopolymer Synthesis Center, and her staff for synthesis of the melittin peptides and for performing analytical HPLC measurements. We thank Catherine Sarisky and Dr. Scott Ross for help with the 2D NMR experiments. We are grateful to Yi Tang and Dr. Ilya Koltover for helpful discussions and for providing 5,5,5-trifluoroleucine and synthetic intermediates used in these studies. This work was supported by the U.S. Army Research Office and by a gift from the Dow Chemical Company. A.N. thanks the National Institutes of Health for a postdoctoral fellowship.", revision_no = "13", abstract = "We have investigated the effect of trifluoroleucine substitution on the membrane-binding and tetramerization behavior of melittin. Analogues were synthesized in which Leu 9, Leu 13, and all four intrinsic leucine residues of melittin were replaced by 5,5,5-trifluoroleucine. Both the mono- and tetra-substituted melittins were found to exhibit stronger self-association and enhanced affinity for lipid bilayer membranes, compared to the wild-type peptide. The extent of the observed effects depends on the site of introduction of trifluoroleucine and, in the case of substitution at position 13, on the stereochemistry of the trifluoroleucine side chain. Analysis of the membrane association isotherms is consistent with aggregation of fluorinated melittins within the lipid bilayer. These results suggest that fluorocarbon−hydrocarbon separation, in addition to an increase in hydrophobic character, contributes to enhanced membrane binding.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53353, title ="Identification of an expanded set of translationally active methionine analogues in Escherichia coli", author = "Kiick, K. L. and Weberskirch, R.", journal = "FEBS Letters", volume = "502", number = "1-2", pages = "25-30", month = "July", year = "2001", doi = "10.1016/S0014-5793(01)02657-6", issn = "0014-5793", url = "https://resolver.caltech.edu/CaltechAUTHORS:KIIfl2001", note = "© 2001 Elsevier. \n\nReceived 6 June 2001; accepted 13 June 2001. First published online 9 July 2001. \n\nThis work was supported by grants from the Polymers and Genetics Programs of the U.S. National Science Foundation and from the U.S. Army Research Office. We are grateful to J.C.M. van Hest and to H. Blackwell for synthesis of methionine analogues and to H. Jakubowski and Y. Mechulam for donation of plasmids encoding MetRS. K.L.K. thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship.", revision_no = "13", abstract = "Amino acid incorporation into proteins in vivo is controlled most stringently by the aminoacyl-tRNA synthetases. Here we report the incorporation of several new methionine analogues into protein by increasing the rate of their activation by the methionyl-tRNA synthetase (MetRS) of Escherichia coli. cis-Crotylglycine (4), 2-aminoheptanoic acid (7), norvaline (8), 2-butynylglycine (11), and allylglycine (12) will each support protein synthesis in methionine-depleted cultures of E. coli when MetRS is overexpressed and the medium is supplemented with the analogue at millimolar concentrations. These investigations suggest important opportunities for protein engineering, as expansion of the translational apparatus toward other amino acid analogues by similar strategies should also be possible.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53958, title ="Fluorinated Coiled-Coil Proteins Prepared In Vivo Display Enhanced Thermal and Chemical Stability", author = "Tang, Yi and Ghirlanda, Giovanna", journal = "Angewandte Chemie International Edition", volume = "40", number = "8", pages = "1494-1496", month = "April", year = "2001", doi = "10.1002/1521-3773(20010417)40:8<1494::AID-ANIE1494>3.0.CO;2-X", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150121-143801079", note = "Issue published online: 17 APR 2001. Article first published online: 17 APR 2001. Manuscript Received: 2 JAN 2001.\n\nThis work was supported by a grant from the U.S. Army Research Office. Y. Tang is supported by a Whitaker Graduate Research Fellowship. We thank Dr. Gary Hathaway for performing matrix-assisted laser desorption/ionization analyses.", revision_no = "8", abstract = "Fluorination of the hydrophobic core of a coiled-coil protein significantly improved its stability toward thermal and chemical denaturation. 5′,5′,5′-Trifluoroleucine (2) was efficiently incorporated into a leucine-zipper protein in place of leucine (1) during E. coli biosynthesis. The fluorinated variant maintained stable secondary and tertiary structures under conditions that caused denaturation of the “wild-type” protein.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53982, title ="Stabilization of Coiled-Coil Peptide Domains by Introduction of Trifluoroleucine", author = "Tang, Yi and Ghirlanda, Giovanna", journal = "Biochemistry", volume = "40", number = "9", pages = "2790-2796", month = "March", year = "2001", doi = "10.1021/bi0022588", issn = "0006-2960", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150122-091952579", note = "Copyright © 2001 American Chemical Society.\nPublished In Issue March 06, 2001. Publication Date (Web): February 7, 2001. Received September 26, 2000. Revised Manuscript Received December 22, 2000.\n\nWe thank Dr. James D. Lear for writing the Igor Pro\nprocedures.. This work was supported by a grant from the U.S. Army Research Office to D.A.T. and by NIH Grant GM54616 to W.F.D. Y. Tang is grateful for a graduate research fellowship from the Whitaker Foundation.", revision_no = "9", abstract = "Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 °C at 30 μM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5−1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor−ligand interactions.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53371, title ="Two-Dimensional Order in β-Sheet Peptide Monolayers", author = "Rapaport, Hanna and Kjaer, Kristian", journal = "Journal of the American Chemical Society", volume = "122", number = "50", pages = "12523-12529", month = "December", year = "2000", doi = "10.1021/ja002238t", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:RAPjacs2000", note = "Copyright © 2000 American Chemical Society. \n\nReceived June 22, 2000. Publication Date (Web): November 30, 2000. \n\nWe thank Dr. Suzanna Horvath for peptide synthesis, Dr. George Rossman for help in ATR infrared spectroscopy, and Dr. William Goddard and Dr. Tahir Cagin for providing the CERIUS2 applications. This work was supported by the United States−Israel Binational Science Foundation, the U.S. National Science Foundation, the Kimmelmann Center, the DanSync program of the Danish Natural Science Research Council, and the European Community under TMR-Contract ERBFMGECT950059. We thank HASYLAB for synchrotron beamtime.", revision_no = "9", abstract = "Amphiphilic peptides comprising alternating hydrophilic and hydrophobic amino acid residues were designed to form super-secondary structures composed of self-assembled β-strands as monolayers at the air−water interface. Insights provided by in situ grazing-incidence X-ray diffraction (GIXD), surface pressure vs area isotherms, and Fourier transform infrared spectroscopy allow structural characterization of the assembled nanostructures and rational correlation with the peptide sequence. Peptides seven to seventeen amino acids in length were found to form crystalline arrays with coherence lengths in the range of 100 to 1000 Å. Two-dimensional registry of the self-assembled peptides was induced by placement of proline residues at the peptide termini. The films were found to intercalate ordered arrays of ions between juxtaposed β-sheet ribbons to generate peptide−ion composite phases.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53715, title ="Effects of Temperature and Pressure on the Aggregation Properties of an Engineered Elastin Model Polypeptide in Aqueous Solution", author = "Tamura, T. and Yamaoka, T.", journal = "Biomacromolecules", volume = "1", number = "4", pages = "552-555", month = "December", year = "2000", doi = "10.1021/bm005606u", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-121449864", note = "© 2000 American Chemical Society. \n\nPublished In Issue December 12, 2000. Publication Date (Web): November 1, 2000. Received September 5, 2000. Revised Manuscript Received October 10, 2000.", revision_no = "8", abstract = "The pressure and temperature dependence of the cloud point transition of an aqueous solution of an elastin-like polypeptide (MGLDGSMG(VPGIG)_(40)VPLE), prepared by bacterial expression of the corresponding artificial gene, was measured. A temperature−pressure diagram was constructed over a wide range of conditions. The (VPGIG)_(40) solution exhibited a well-defined pressure-induced cloudpoint (P_c), as well as a temperature-induced transition (T_c). From near atmospheric pressure up to 100 MPa, T_c increased with increasing pressure, but decreased with further increases in pressure above 200 MPa. The maximum T_c was reached at 100−200 MPa. Between 10 and 25 °C, the Pc decreased with increasing temperature, and a broad maximum in P_c was observed in the range −10 to 0 °C. These results are compared with our previous results on synthetic thermoresponsive vinyl polymers. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53360, title ="Protein Engineering by In Vivo Incorporation of Non-Natural Amino Acids: Control of Incorporation of Methionine Analogues by Methionyl-tRNA Synthetase", author = "Kiick, Kristi L. and Tirrell, David A.", journal = "Tetrahedron", volume = "56", number = "48", pages = "9487-9493", month = "November", year = "2000", doi = "10.1016/S0040-4020(00)00833-4", issn = "0040-4020", url = "https://resolver.caltech.edu/CaltechAUTHORS:KIIt2000", note = "© 2000 Elsevier. \n\nReceived 22 May 2000; accepted 2 August 2000. \n\nThis work was supported by grants from the Polymers and Genetics Programs of the U.S. National Science Foundation and from the U.S. Army Research Office. We are grateful to J. C. M. van Hest, R. Weberskirch, and H. E. Schoemaker for synthesis of the methionine analogues, to H. Jakubowski for donation of the plasmid pGG3, and to R. Alexander for helpful discussions regarding activation assays. K. L. K thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship.", revision_no = "10", abstract = "The incorporation of non-natural amino acids is an important strategy for engineering novel chemical and physical properties into natural and artificial proteins. The incorporation of amino acids into proteins in vivo is controlled in large part by the aminoacyl-tRNA synthetases (AARS). We have measured kinetic constants for in vitro activation of a set of methionine analogues by methionyl-tRNA synthetase (MetRS) via the ATP–PPi exchange reaction. Activation of methionine analogues in vitro correlates well with the ability of these analogues to support protein synthesis in vivo, substantiating the critical role of the AARS in controlling the incorporation of non-natural amino acids into proteins. Methionine analogues with k_(cat)/K_m values 2000-fold lower than those for methionine can support synthesis of a typical target protein (mDHFR) under standard conditions of protein expression. The kinetic constants correlate well with observed protein yields from a conventional bacterial expression host, indicating that the MetRS activity of the host can control the level of protein synthesis under certain conditions. Furthermore, increasing the MetRS activity of the bacterial host results in increased protein synthesis in media supplemented with the methionine analogues homoallylglycine and norleucine. These results suggest new strategies for incorporation of non-natural amino acids via manipulation of the AARS activity of a bacterial host.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53716, title ="Thermal and Structural Properties of Biologically Derived Monodisperse Hairy-Rod Polymers", author = "Yu, Seung M. and Tirrell, David A.", journal = "Biomacromolecules", volume = "1", number = "3", pages = "310-312", month = "September", year = "2000", doi = "10.1021/bm000039w", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-121449953", note = "Copyright © 2000 American Chemical Society.\nPublished In Issue September 12, 2000. Publication Date (Web): August 18, 2000. Received May 10, 2000.\n\nAcknowledgment. This work was supported by the National Science Foundation Materials Research Science and Engineering Center at the University of Massachusetts.\n\nSupporting Information Available. Text giving experimental\ndetails of the synthesis of PHBG and figures showing\nX-ray powder diffraction patterns for PHBG-4.", revision_no = "11", abstract = "Monodisperse derivatives of poly(γ-4-(hexadecyloxy)benzyl α,l-glutamate) (PHBG-X, X = 3 or 4) with backbone sequence GluAsp(Glu_(17)Asp)_xGluGlu were prepared by reaction of 4-(hexadecyloxy)phenyldiazomethane with the corresponding monodisperse poly(α,l-glutamate) (PLGA) derivatives (PLGA-X, X = 3 or 4). PHBG-3 and -4 exhibited strong endotherms near 45 °C and weak endotherms near 86 °C when analyzed by differential scanning calorimetry. X-ray diffraction suggested that these polymers aggregate to form layerlike solid structures at room temperature, with extended alkyl side chains forming paraffinlike crystallites. Most of the side chain order disappears at the first melting transition; however, the layerlike structure remains. Both polymers are isotropic above the second melting transition; no ordered melts were observed at higher temperatures, possibly due to the small aspect ratios of PHBG-3 and -4. In contrast, polydisperse poly(γ-4-(hexadecyloxy)benzyl α,l-glutamate) (PDI = 1.2, DP = 98) (PHBG-P1), prepared from commercial PLGA, formed liquid crystalline (LC) phases between 97 and 105 °C. ", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53844, title ="Nanometer-Scale Smectic Ordering of Genetically Engineered Rodlike Polymers:\u2009 Synthesis and Characterization of Monodisperse Derivatives of Poly(γ-benzyl α,L-glutamate)", author = "Yu, Seungju M. and Soto, Carissa M.", journal = "Journal of the American Chemical Society", volume = "122", number = "28", pages = "6552-6559", month = "July", year = "2000", doi = "10.1021/ja000465p", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150116-151446955", note = "Copyright © 2000 American Chemical Society.\nPublished In Issue July 19, 2000. Publication Date (Web): June 29, 2000. Received February 7, 2000.\n\nAcknowledgment. This work was supported by the National\nScience Foundation Materials Research Science and Engineering\nCenter at the University of Massachusetts, and by grants from\nthe Polymers and Genetics Programs of the NSF.", revision_no = "9", abstract = "Macromolecules with precisely defined architectures on the nanometer scale are ideal building blocks for self-assembled materials and nanometer-scale devices. By utilizing recombinant DNA technology and bacterial protein expression, we have prepared a set of rodlike artificial proteins, Glu(OBzl)Asp(OBzl)[Glu(OBzl)_(17)Asp(OBzl)]_X-Glu(OBzl)Glu(OBzl) (PBLG-X, X = 3−6, 1), which serve as monodisperse analogues of poly(γ-benzyl α,l-glutamate) (PBLG), with variation of rod length from 8.7 to 17 nm. Synthesis was accomplished by (i) bacterial production of a precursor polypeptide GluAsp(Glu_(17)Asp)_XGluGlu (PLGA-X, X = 3−6), as a fusion to mouse dihydrofolate reductase (DHFR), (ii) CNBr digestion of the expressed protein to liberate PLGA-X, and (iii) side chain benzylation of PLGA-X via treatment with phenyldiazomethane. The growth rates of cultures transformed with the recombinant genes, and the yields of protein expressed therein, depended on the number of Glu_(17)Asp repeats appended to the fusion partner, decreasing as the number of repeats increased. PBLG-3, with the lowest aspect ratio (6.9), did not exhibit liquid crystalline (LC) behavior while each of the other polymers formed lyotropic LC phases. The phase transition behavior of PBLG-X was distinctly different from that of conventional polydisperse PBLG; when the concentration of the polymer solution was gradually increased, the transition from the isotropic to the liquid crystalline state occurred uniformly throughout the sample without formation of distinct birefringent droplets. Solution-cast films of PBLG-4 and PBLG-5 formed smectic supramolecular architectures with layer spacings (11.4 and 14.0 nm, respectively) precisely determined by the length of the monodisperse rods. Smectic ordering was observed only in films cast from mixtures of chloroform (97%) and trifluoroacetic acid (3%). In films prepared from dioxane, both polydisperse and monodisperse PBLGs adopted columnar order without formation of smectic layers.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53375, title ="Polymer-induced leakage of cations from dioleoyl phosphatidylcholine and phosphatidylglycerol liposomes", author = "Thomas, James L. and Tirrell, David A.", journal = "Journal of Controlled Release", volume = "67", number = "2-3", pages = "203-209", month = "July", year = "2000", doi = "10.1016/S0168-3659(00)00209-1", issn = "0168-3659", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-111319852", note = "© 2000 Elsevier Science B.V. All rights reserved.\n\nReceived 24 September 1999; accepted 19 January 2000.", revision_no = "10", abstract = "The amphipathic polyacid, poly(2-ethylacrylic acid) (PEAA) has recently been shown to form fluctuating channels in patch-clamp measurements of phospholipid bilayers [J.C. Chung, D.J. Gross, J.L. Thomas, D.A. Tirrell, L.R. Opsahl-Ong, Macromolecules 29 (1996) 4636–4641.]. To explore this phenomenon further, we have quantified the PEAA-mediated pH-dependent release of sodium and calcium ions from phospholipid vesicles. Permeability to calcium increases linearly with polymer concentration and exponentially with decreasing pH. Permeabilization of negatively charged phosphatidylglycerol (PG) liposomes occurs to a similar extent and with a similar pH dependence to that of zwitterionic phosphatidylcholine (PC) liposomes, implying that a charge neutral species is responsible for the leakage. The pH dependence of leakage shows that the cooperative protonation of from three to five carboxylate anions is required for permeabilization. Such neutralization could result in a neutral segment of polymer chain of sufficient length to span the bilayer.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53956, title ="Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase Activity of a Bacterial Expression Host", author = "Kiick, Kristi L. and van Hest, Jan C. M.", journal = "Angewandte Chemie International Edition", volume = "39", number = "12", pages = "2148-2152", month = "June", year = "2000", doi = "10.1002/1521-3773(20000616)39:12<2148::AID-ANIE2148>3.0.CO;2-7", issn = "1433-7851", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150121-143039773", note = "Issue published online: 14 JUN 2000. Article first published online: 14 JUN 2000. Manuscript Received: 26 JAN 2000.\n\nScott Ross was helpful in conducting the 1D TOCSY NMR experiments and Pratip Bhattachary is thanked for assistance in other NMR experiments. We are grateful to Yves Mechulam for a sample of plasmid pBSM547W305F and to Hieronim Jakubowski of UMDNJ-New Jersey Medical School, Newark, New Jersey, for plasmid pGG3. K.L.K. thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship. This work was supported by grants from the Polymers and Genetics Programs of the National Science Foundation and from the U.S. Army Research Office.", revision_no = "9", abstract = "The incorporation of amino acids into proteins in vivo is controlled by the aminoacyl-tRNA synthetases. The successful incorporation of a nonnatural amino acid, trans-crotylglycine (Tcg), into a protein has now been achieved by increasing the methionyl-tRNA synthetase activity of a bacterial expression host (see scheme). The incorporation of Tcg into proteins creates new opportunities for macromolecular synthesis through genetic engineering, due to the rich chemistry of the olefinic side chain.", } @book_section {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53733, title ="Characterization of poly(2-\u200bethylacrylic acid) for use in drug delivery", author = "Linhardt, Jeffrey G. and Tirrell, David A.", number = "752", pages = "243-252", month = "May", year = "2000", doi = "10.1021/bk-2000-0752.ch024", isbn = "9780841236257", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-142433283", note = "© 2000 American Chemical Society. \n\nThis work was supported by a grant from INEX Pharmaceuticals, Inc.", revision_no = "17", abstract = "The pH-dependent conformational transition of poly(2-ethylacrylic acid) [PEAA] and its\ninteraction with phosphotidylcholine bilayer membranes were investigated. The conformational transition\nwas monitored with fluorescence spectroscopy using pyrene as a probe; the location and breadth of the\nconformational transition was shown to be dependent on the molecular weight and polydispersity of the\nsample, respectively. The pH-dependent destabilization and fusion of extruded large unilamellar vesicles\n(LWs) by PEAA was characterized by optical density measurements, transmission electron microscopy,\nand lipid mixing and contents release assays. Reduction of either the chain length or the polymer\nconcentration caused the fusion and contents release events to shift to lower pH values. Release of\nentrapped calcein was observed at pH values ca. 1 unit higher than those found to cause membrane fusion.\nDecreased levels of fusion were observed when the concentration of PEAA was lower than that of the\nlipid; however, quantitative release of encapsulated calcein could be effected at very low polymer\nconcentrations (-3% wlw PEAA/lipid).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53717, title ="Engineering the Extracellular Matrix:\u2009 A Novel Approach to Polymeric Biomaterials. I. Control of the Physical Properties of Artificial Protein Matrices Designed to Support Adhesion of Vascular Endothelial Cells", author = "Welsh, Eric R. and Tirrell, David A.", journal = "Biomacromolecules", volume = "1", number = "1", pages = "23-30", month = "March", year = "2000", doi = "10.1021/bm0002914", issn = "1525-7797", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150114-121450041", note = "Copyright © 2000 American Chemical Society.\nPublished In Issue March 14, 2000. Publication Date (Web): February 4, 2000. Received January 17, 2000.\n\nAcknowledgment. This work was supported by National\nInstitutes of Health grant HL59987.", revision_no = "8", abstract = "Methods of genetic engineering were applied to the design and biosynthesis of three extracellular matrix protein analogues constructed from identical elastin- and fibronectin-derived repeating units but characterized by different molecular weights in the range of 14\u2009000 to 59\u2009000. Expression levels were enhanced by the serendipitous choice of an N-terminal fusion sequence such that gram-scale syntheses were achieved for each protein. Purification protocols were developed that resulted in proteins of high purity and correct sequence, as determined by amino acid analysis, NMR spectroscopy, and lower critical solution temperature (LCST). Glutaraldehyde was shown to insolubilize the otherwise soluble proteins in a concentration-dependent manner. Tensile moduli of cross-linked protein films were measured and found to be inversely related to the molecular weights of the engineered proteins, which in each case corresponds to the theoretical molecular weight between cross-links. At the highest cross-link density (lowest molecular weight) the elastic modulus was similar to that of native elastin.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53379, title ="Efficient Incorporation of Unsaturated Methionine Analogues into Proteins in Vivo", author = "van Hest, Jan C. M. and Kiick, Kristi L.", journal = "Journal of the American Chemical Society", volume = "122", number = "7", pages = "1282-1288", month = "February", year = "2000", doi = "10.1021/ja992749j", issn = "0002-7863", url = "https://resolver.caltech.edu/CaltechAUTHORS:HESjacs2000", note = "© 2000 American Chemical Society. \n\nReceived August 2, 1999. Publication Date (Web): February 3, 2000. \n\nThis work was supported by a grant from the Polymers and Genetics Programs of the U. S. National Science Foundation. The Netherlands Organization for Scientific Research (NWO) and DSM Research are acknowledged for unrestricted grants in support of our research. We are grateful to H. Jakubowski for plasmid pGG3. K.L.K thanks the U.S. Department of Defense for a National Defense Science and Engineering Graduate Fellowship.", revision_no = "10", abstract = "A set of eight methionine analogues was assayed for translational activity in Escherichia coli. Norvaline and norleucine, which are commercially available, were assayed along with 2-amino-5-hexenoic acid (2), 2-amino-5-hexynoic acid (3), cis-2-amino-4-hexenoic acid (4), trans-2-amino-4-hexenoic acid (5), 6,6,6-trifluoro-2-aminohexanoic acid (6), and 2-aminoheptanoic acid (7), each of which was prepared by alkylation of diethyl acetamidomalonate with the appropriate tosylate, followed by hydrolysis. The E. coli methionine auxotroph CAG18491, transformed with plasmids pREP4 and pQE15, was used as the expression host, and translational activity was assayed by determination of the capacity of the analogue to support synthesis of the test protein dihydrofolate reductase (DHFR) in the absence of added methionine. The importance of amino acid side chain length was illustrated by the fact that neither norvaline (8) nor 7 showed translational activity, in contrast to norleucine (9), which does support protein synthesis under the assay conditions. The internal alkene functions of 4 and 5 prevented incorporation of these analogues into test protein, and the fluorinated analogue 6 yielded no evidence of translational activity. The terminally unsaturated compounds 2 and 3, however, proved to be excellent methionine surrogates:\u2009 ^1H NMR spectroscopy, amino acid analysis, and N-terminal sequencing indicated ∼85% substitution of methionine by 2, while 3 showed 90−100% replacement. Both analogues also function efficiently in the initiation step of protein synthesis, as shown by their near-quantitative occupancy of the N-terminal amino acid site in DHFR. Enzyme kinetics assays were conducted to determine the rate of activation of each of the methionine analogues by methionyl tRNA synthetase (MetRS); results of the in vitro assays corroborate the in vivo incorporation results, suggesting that success or failure of analogue incorporation in vivo is controlled by MetRS.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53378, title ="Efficient introduction of aryl bromide functionality into proteins in vivo", author = "Sharma, Nandita and Furter, Rolf", journal = "FEBS Letters", volume = "467", number = "1", pages = "37-40", month = "February", year = "2000", doi = "10.1016/S0014-5793(00)01120-0", issn = "0014-5793", url = "https://resolver.caltech.edu/CaltechAUTHORS:SHAfl2000", note = " © 2000 Federation of European Biochemical Societies. \n\nReceived 22 November 1999; received in revised form 10 January 2000. \n\nThis work was supported by a grant from the U.S. Army Research Office.", revision_no = "10", abstract = "Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein-based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para-bromophenylalanine (p-Br-phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl-tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p-Br-phe-supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p-Br-phe; variation in the relative amounts of phe and p-Br-phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20–25 mg/l were obtained from cultures supplemented with p-Br-phe; this corresponds to about two-thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post-translational derivatization of brominated proteins via metal-catalyzed coupling reactions. In addition, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/54249, title ="Les protéines artificielles", author = "Michon, Thiery and Tirrell, David A.", journal = "Biofutur", volume = "2000", number = "197", pages = "34-38", month = "February", year = "2000", doi = "10.1016/S0294-3506(00)88138-7", issn = "0294-3506", url = "https://resolver.caltech.edu/CaltechAUTHORS:20150209-155252856", note = "Copyright © 2000 Published by Elsevier Masson SAS.\nAvailable online 20 April 2000.", revision_no = "12", abstract = "Certaines substances naturelles, la soie d’araignee par exemple, manifestent une extraordinaire resistance a la traction. En combinant les motifs proteiques qui les composent avec des sequences provenant d’autres molecules, la biotechnologie permet desormais de creer des biomateriaux inedits.", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53381, title ="pH-Induced Fusion and Lysis of Phosphatidylcholine Vesicles by the Hydrophobic Polyelectrolyte Poly(2-ethylacrylic Acid)", author = "Linhardt, Jeffrey G. and Tirrell, David A.", journal = "Langmuir", volume = "16", number = "1", pages = "122-127", month = "January", year = "2000", doi = "10.1021/la9906267", issn = "0743-7463", url = "https://resolver.caltech.edu/CaltechAUTHORS:LINl2000", note = "Copyright © 2000 American Chemical Society. \n\nReceived May 24, 1999. Publication Date (Web): December 10, 1999. \n\nThe authors thank Howard Bowman, Wendy Naimark, Jan van Hest, and James Thomas for helpful discussions, as well as David Flanagan for review of this manuscript and discussions. This work was supported by a grant from INEX Pharmaceuticals, Inc. \n\nPart of the Special Issue “Clifford A. Bunton: From Reaction Mechanisms to Association Colloids; Crucial Contributions to Physical Organic Chemistry”. This paper is dedicated to Professor Clifford A. Bunton on the occasion of his eightieth birthday.", revision_no = "9", abstract = "Poly(2-ethylacrylic acid) [PEAA] was shown to induce fusion of phosphatidylcholine bilayer membranes under mildly acidic conditions. The pH-dependent destabilization and fusion of extruded large unilamellar vesicles (LUVs) by PEAA was characterized by optical density measurements, transmission electron microscopy, and lipid-mixing and contents-release assays. Reduction of either the chain length or the polymer concentration caused the fusion and contents-release events to shift to lower pH values. Release of entrapped calcein was observed at pH values approximately 1 unit higher than those found to cause membrane fusion. Decreased levels of fusion were observed when the concentration of PEAA was lower than that of the lipid; however, quantitative release of encapsulated calcein could be effected at very low polymer concentrations (∼3% w/w PEAA/lipid).", } @article {CaltechAUTHORS_https://authors.library.caltech.edu/id/eprint/53382, title ="Structure of poly(γ-benzyl-L-glutamate) monolayers at the gas–water interface: A Brewster angle microscopy and x-ray scattering study", author = "Fukuto, Masafumi and Heilmann, Ralf K.", journal = "Journal of Chemical Physics", volume = "111", number = "21", pages = "9761", month = "December", year = "1999", doi = "10.1063/1.480312", issn = "0021-9606", url = "https://resolver.caltech.edu/CaltechAUTHORS:FUKjcp1999", note = "© 1999 American Institute of Physics. \n\nReceived 14 June 1999; accepted 9 September 1999. \n\nThe authors thank H. Tostmann, O. G. Shpyrko, K. Penanen, E. DiMasi, B. M. Ocko, and M. Deutsch for helpful discussions regarding the off-specular diffuse scattering technique. The Harvard contribution to this work was supported by Grant No. NSF-DMR-98-72817. Work at the University of Massachusetts was supported by Grant No. NSF-DMR-95-10031. The x-ray experiments were carried out on the Harvard/BNL liquid surface spectrometer at Beamline X22B in the National Synchrotron Light Source, Brookhaven National Laboratory. This facility is supported by No. DEAC02-76CH00016.", revision_no = "8", abstract = "This paper reports Brewster angle microscopy (BAM), x-ray specular reflectivity (XR), grazing incidence diffraction(GID) and off-specular diffuse scattering (XOSDS) measurements of Langmuir monolayers formed on water by both mono- and polydisperse samples of α-helical poly(γ-benzyl L-glutamate) (PBLG) as a function of area/monomer A. The microscopic behavior does not exhibit any discernible effects due to differing dispersity. At low surface densities (A>∼21Å^2/monomer,surface pressure Π=0), BAM images reveal partial surface coverage by solidlike monolayer islands. GID measurements show an interhelix peak corresponding to a local parallel alignment of rodlike PBLG molecules, indicating their tendency to aggregate laterally without external pressure. Compression to A<21Å^2/monomer first leads to full and uniform surface coverage by the monolayer, followed by a steep rise in Π that is accompanied by a decrease in the interhelix distance. Further compression results in a plateau of constant Π in the Π-A isotherm (∼11.5