David A. Tirrell - US grants

The Industry/University Cooperative Research Center at the University of Massachusetts, Amherst is now a self-sufficient research group addressing basic polymer science. This project is a collaborative study between the Center for University of Massachusetts - Industry Research on Polymers and Mount Holyoke College. The project is studying the development and examination of synthetic vesicle systems which "sense" their environment via conformational changes of membranes-bound synthetic polymers.

This award will support the participation of six U.S. scientists in a joint seminar entitled "Advanced Materials Based on Macromolecules," to be held under the auspices of the U.S.- Japan Cooperative Science Program. The focus of the meeting will be on recent advances in polymer synthesis that provide new levels of control of polymer structure and that underlie future developments in optical, electronic, biomedical, and high-strength materials. Previous U.S.-Japan seminars in areas of polymer synthesis served as opportunities for research groups in the two countries to share ideas and information, and in several cases led to cooperative research projects and exchange visits. Similar results are expected from the present meeting. Specific topics to be discussed at the seminar include recent advances in the control of polymer chain structure, advances in optical properties of polymeric materials and in conducting polymers, liquid crystalline polymers, radiation-sensitive polymers, polymers in biology and medicine, and polymers in composite materials. The seminar will be held October 29 - November 2, 1990, in Tokyo, Japan. The co-organizers are Professor David A. Tirrell, Department of Polymer Science and Engineering, University of Massachusetts at Amherst, and Professor Eishun Tsuchida, Department of Polymer Chemistry, Waseda University, Tokyo, Japan. Advanced materials underlie current and projected developments in electronics, communications, aerospace, automotive, and health care industries, among others. Considerable research on advanced materials based on macromolecules is being carried out in the United States and Japan. This seminar will highlight recent advances in polymer synthesis in the two countries that point the way to future materials development.

9510031 Tirrell This project will develop methods for the preparation of architecturally well-defined artificial proteins carrying reactive olefinic, dienyl, acetylenic and pyrrolyl functional groups. A key aspect of the project is the use of artificial genes to direct the synthesis of uniform polymer chains, which will acquire reactive functionality either directly (through the incorporation of unnatural amino acids), or indirectly (through post-translational modification reactions). In either approach, the fidelity of bacterial protein synthesis will be exploited to produce polymeric materials characterized by precise control of chain length, sequence, and stereochemistry. The direct approach will explore the translational activity of six artificial amino acids (and several variants thereof) related to isoleucine, phenylalanine, histidine or tyrosine. There is increasing evidence that the bacterial protein synthesis machinery can utilize a braoder range of amino acids than the twenty that are normally encoded by messenger RNA templates. The amino acid analogues chosen for this study are of particular interest owing to two facts: i). none of the natural amino acids carries olefinic, dienyl, acetylenic or pyrrolyl groups, and ii). such functional groups can be used to endow polymeric materials with interesting and potentially useful chemical and electrochemical properties. The approach to be used for direct incorporation involves: i). synthesis of an artificial gene encoding the sequence of interest, with the natural analogue encoded in place of the target unnatural amino acid, ii). cloning of the artificial gene in an appropriate auxotrophic bacterial strain, and iii). induction of target protein synthesis in a medium containing the unnatural amino acid (and generally depleted of the natural analogue). This strategy has been demonstrated successfully with several unnatural amino acids, including selenomethionine, p-fluorophenylalanine, 3- thienylalanine, trifluoroleucine, azetidinecarboxylic acid, dehydroproline, and thiaproline. Complementary strategy involving post-translational modification will also be explored. In this approach, protein biosynthesis will be used to prepare appropriately designed "base polymers," which will be modified with reagents carrying olefinic, dienyl, acetylenic or pyrrolyl groups. Polymers prepared by either route will be subjected to careful analyses of molecular structure, supramolecular organization, and functional properties. Particular attention will be given to the prospects for creating electrochemically switchable protein films containing pyrrolyl side chains. Such films would be of potential practical use as components of sensors, actuators and controlled delivery devices. %%% This research aims to carry out the synthesis of amino acid polymers with highly controlled structures, through the use of artificial genes. The polymers selected do not occur in nature and will be designed to have interesting electrical and other properties. ***

The Materials Research Laboratory (MRL) at the University of Massachusetts - Amherst supports interactive, interdisciplinary research in polymer science and engineering organized into three major thrust areas. Thrust research on pattern formation in polymer systems has evolved from earlier work in the MRL on polymer blends. It focusses on structure and pattern formation in multiphase polymer systems, including both blends and liquid crystals. Researchers in the thrust area on polymers in restricted geometries are synthesizing and characterizing novel polymeric films and surfaces, investigating their corresponding properties, and studying polymer adsorption processes. A new thrust on ordered polymeric solids is devoted to novel approaches to polymer synthesis, characterization of new periodic polymer morphologies, processing and property prediction. A major emphasis is placed on novel polymers of precisely defined structure produced by incorporating biochemical techniques into the synthetic procedures. The MRL also supports the development, maintenance and operation of central research facilities for characterization, morphology and rheology determination, spectroscopy, and computation. It also provides seed funding for exploratory research and new initiatives in materials research, and supports programs to foster increased participation by minorities and to provide experience in polymer research for undergraduates. The research program currently involves 19 faculty members from four departments, 3 postdoctoral research associates, 19 graduate students and 15 undergraduates, and supporting technical and clerical staff. The MRL at the University of Massachusetts, Amherst is directed by Professor David A. Tirrell.

This is a proposal a 300 MHz solid state NMR spectrometer that will be used for research which addresses important topics in the fields of biomolecular structure and surface and polymer chemistry. The biophysical projects will investigate the structure and mechanism of membrane proteins, with the aim of understanding the role of conformational changes in signal transduction and the role of membrane inserton in the gating of channels. The instrument will also enable high field studies which will compoement an existing zero field NMR program to investigate the chemistry of catalytic surfaces. Finally, this instrument will enhance a strong program in polymer science, with projects ranging from the investigation of structure and dynamics in polymers with powerful new two dimensional solid state NMR techniques ot the characterization of an exciting new class of materials, biopolymers. This is an ideal environment for expanding the application of solid state NMR to answer biological questions, because of the confluence of several soid state NMR spectroscopists and numerous investigators in the bioligical science who already participate in an interdisciplinary graduate program in molecular and cellular biology.

CTS-9512485 Tsapatsis U of Massachusetts A Powder X-ray Diffractometer (PXRD) will be procured for research in material characterization. The PXRD will have capabilities for in situ high temperature, controlled environment and thin film XRD. Uses for the equipment include characterization of synthetic zeolites, polymeric materials and amphiphilic association structures, as well as identification of layered silicates and other minerals in geological samples. It will support various research projects in advanced materials processing and manufacturing. The PXRD will also be used for training of graduate students.

The long-term objective of this work is the development of new genetically engineered materials to be used in the fabrication of vascular grafts characterized by improving long-term patency. The approach is predicated on the following hypotheses: i). that existing synthetic vascular graft materials - specifically expanded polytetrafluoroethylene (ePTFE) and Dacron - are not optimal, either in terms of surface chemistry or with respect to mechanical properties, ii). that the mechanisms of healing of synthetic grafts are controlled at least in part by cellular interactions with the graft surface or with macromolecules, including plasma proteins or extracellular matrix (ECM) proteins, deposited on that surface, iii). that a measure of control of cellular behavior at the graft surface can be gained by presentation of ligands for specific cell- surface receptors, and iv). that engineered variants of ECM proteins will allow control, both of the key mechanical properties of the graft, and of the presentation of ligands at the graft surface. The project will include: i). microbial expression of artificial genes encoding artificial ECM proteins that incorporate specific cell-binding and crosslinking domains, ii). fabrication and mechanical characterization of crosslinked films prepared from these proteins, iii). determination of the adhesivity of these surfaces with respect to endothelial cells cultured under physiologically relevant shear stresses, and iv). analysis of the behavior of endothelial cell monolayers cultured on artificial extracellular matrices.

The objective of this project is to design and synthesize a new class of protein biomaterials for use in vascular graft applications. The proteins consist of repeats of elastin derived polypeptides with various cell binding peptides. They will be produced by expressing artificial genes in bacteria. The molecular structure of the materials will be manipulated via peptide sequence modifications and crosslinking methods in order to optimize mechanical and cell interaction properties. Finally, the attachment, spreading and function of vascular endothelial and smooth muscle cells on these materials will be evaluated under both static and perfused culture conditions, as a function of molecular properties.

9877039

Abstract

This project involves the acquisition of a 600 MHz NMR spectrometer for the Instrumentation facilities of the Division of Chemistry and Chemical Engineering of California Institute of Technology. The major purpose of this high field instrument will be for the determination of the structures of complex biological molecules of biosynthetic, semisynthetic and synthetic origin. The research activities of several groups in the Division will benefit greatly from the new spectrometer and in many cases, entirely new projects will be made possible by the new instrument. The 600 MHz spectrometer has the resolution and sensitivity needed to determine structures of complexes in the 5 to 30 kD range. Research for which the instrument is required includes the study of metal-DNA complexes; protein- and peptide-DNA complexes; the structure of DNA intercalators; studies of the hyperthermostability of rubredoxin from P. furiosus; structure and dynamics of novel proteins; artificial proteins; ion channel proteins; and electron transfer reactions in wild-type and mutant netalloproteins. The availability of this instrument will have a major impact on both the research and training activities of the Division.

The statistical nature of step and chain growth polymerization processes ensures that the products of such reactions must be heterogeneous. Conventional p9olymeric materials therefore consist of complex mixtures of chains, often characterized by broad distributions of chain length, sequence and stereochemistry. In many materials applications, this kind of molecular heterogeneity is acceptable, and in some applications it is advantageous, insofar as it may suppress crystallization and preserve desirable properties such as optical clarity, elasticity or ease of processing. On the other hand, synthetic developments that have afforded improved control of macromolecular architecture, such as Ziegler-Natta catalysis and living polymerization, have had profound impact on polymer science and technology.
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Over the past decade, several laboratories - including the PI's - have exploited the capacity of the protein biosynthetic apparatus of bacterial cells to make new macromolecular materials characterized by essentially complete control of molecular architecture. The most important advantages of this method appear to lie in two areas: i). In the integration of material properties with biological function, and ii). In the design and fabrication of small-scale structures (sometimes called nanostructures), in which uniformity of molecular architecture is critical. This proposal addresses the latter objective, with a specific focus on the assembly behavior of systems containing monodisperse macromolecular rods related to poly (g-benzyl a,L-glutamate) (PBLG). This research is co-funded by the Polymers Program in the Division of Materials Research and the Molecular Biochemistry Program in the Division of Molecular and Cellular Biosciences.
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DESCRIPTION (provided by applicant): This project will develop powerful new tools to determine when and where proteins are made in complex cellular systems. Reactive amino acid analogs will be incorporated into cellular proteins and selectively conjugated to probes for visualization, isolation and identification of newly synthesized proteins in both prokaryotic and eukaryotic cells. Most importantly, these approaches can provide both spatial and temporal resolution in analysis of cellular protein synthesis. Cell-selectivity is achieved by controlled expression of mutant aminoacyl-tRNA synthetases;in systems containing multiple cell types, amino acid labeling is confined to cells in which the mutant synthetase is active. This project will explore the use of such methods to elucidate the mechanisms by which bacteria evade the defenses of mammalian hosts, to examine the process of quorum sensing (which is essential to the expression of virulence by bacterial pathogens), and to interrogate protein synthesis in a cell-selective manner in the model organism Caenorhabditis elegans. These studies will establish powerful, general platforms for systems-level characterization of biological phenomena ranging from development to the treatment of disease. PUBLIC HEALTH RELEVANCE: This project will provide new methods to elucidate the mechanisms by which bacteria attempt to evade the defenses of their mammalian hosts, to examine how bacteria communicate with one another to express virulence factors and form antibiotic-resistant biofilms, and to identify the different sets of proteins made by individual cells in living animals. These studies will establish new windows on biological phenomena ranging from growth and development to the treatment of infectious disease.

DESCRIPTION (provided by applicant): The long-term objective of this work is the development of artificial extracellular matrix (aECM) proteins to be used in the fabrication of vascular grafts characterized by improving long-term patency. The approach is predicated on the following hypotheses: I). that existing synthetic vascular graft materials - specifically expanded polytetrafluoroethylene (ePTFE) and Dacron - are not optimal, either in terms of surface chemistry or with respect to mechanical properties, ii). that the mechanisms of healing of synthetic grafts are controlled at least in part by cellular interactions with the graft surface or with macromolecules, including plasma proteins or ECM proteins, deposited on that surface, iii). that a measure of control of cellular behavior at the graft surface can be gained by presentation of ligands for specific cell-surface receptors, and iv). that engineered variants of ECM proteins will allow control, both of the key mechanical properties of the graft, and of the presentation of ligands at the graft surface. The project will include: I). determination of endothelial cell spreading and adhesion behavior on crosslinked aECM proteins, ii). determination of the effect of aECM structure on endothelial cell migration and proliferation, and iii). in vitro analysis of inflammatory and thrombogenic responses to aECM proteins.

Under renewed NSF support, The Center for the Science and Engineering of Materials (CSEM) at Caltech will carry forward its mission as a multifaceted materials research and education center. CSEM combines world-class materials research programs organized as interdisciplinary research groups and seed research projects, with educational programs serving underrepresented minority undergraduate, community college, and high school students across Southern California and also serving the general public via television-based mass media programming.

The Center will address both research and educational aspects of materials science in several areas: i) Macromolecular materials design to produce tailored responses to cellular adhesion and growth; ii) novel ferroelectric photonic materials that enable new freedoms in tuning the dispersion relations for photonic materials and devices and offer a new avenue for scientific progress in using light to understand complex materials behavior; and iii) advanced structural materials based on bulk metallic glass composites, with the potential to enable new amorphous structural materials with strength-to-weight ratio much higher than steel.

CSEM also supports emerging research areas via seed projects, which will focus on i) catalytic materials for chemical storage of hydrogen via methanol production and use in nonpolymer fuel cells based on "superprotonic" solid acids and ii) spintronic and optoelectronic properties of organic semiconductor/ferromagnetic heterostructures with applications in future electronic and quantum computing devices.

With this award from the Chemistry Research Instrumentation and Facilities: Departmental Multiuser Instrument Acquisition (CRIF:MU) Program, the Department of Chemistry and Chemical Engineering at California Institute of Technology will acquire a liquid chromatograph-time-of-flight mass spectrometry (LC-TOF) system. Representatives from the numerous projects that would take advantage of the new LC-TOF are the following: a) identification of the metabolites of libraries of novel cytochromes P450 that are generated by methods of directed evolution; b) identification and quantification of the oligomeric components of aerosols with the aim of understanding their global impact on health and climate; c) identification of the chemical composition of the atmosphere of Titan, the largest moon of Saturn; d) guided organic synthesis where complex bioactive molecules are synthesized by novel, and more efficient pathways; and e) identification and quantification of new bioactive molecules in plants with interesting pharmacological properties.

Liquid chromatography with mass spectrometric detection is an extremely powerful technique used for the separation and analysis of complex mixtures. In addition to providing analytical support for a broad range of exciting scientific problems, the new LC-TOF will play a more important educational role. First is the enhancement of the current educational and research activities of the graduate students and post-docs who will receive mass spectrometric training and gain ready access to this new LC-TOF to implement their research. Second and more importantly, is the enhancement of the learning experiences of the undergraduates who participate in the Summer Undergraduate Research Fellowship and Minority Undergraduate Research Fellowship programs. These programs sponsor talented undergraduates to spend a summer working in a research lab on the Caltech campus, and those whose projects require mass spectroscopy will receive training and hands-on experience at this facility.

With this award from the Chemistry Research Instrumentation and Facilities: Multi User program (CRIF:MU), the Department of Chemistry and Chemical Engineering at California Institute of Technology will acquire an X-ray diffractometer (CCD) with a low-temperature crystal cooling system (LT). It will be utilized in research projects including 1) synthesis of organometallic catalysts for olefin polymerization, fuel production, and value-added chemicals, 2) mechanistic studies of zeolites, 3) mechanistic studies of protein folding as well as redox properties and molecular wire electron transfer of proteins, 4) development of corrole compounds for solar cell development, 5) development of complex macrocycles for biosensor development, 6) development of metathesis catalysts, 7) mechanistic studies of the catalytic processes of metallic centers in natural systems, and 8) "guided" synthesis of complex and biologically relevant organic compounds. The new X-ray diffractometer will be housed in the X-ray Crystallography Lab of the Division of Chemistry and Chemical Engineering in the Beckman Institute at the California Institute of Technology.

The technique of single-crystal X-ray crystallography allows accurate and precise determination of the full three dimensional structure of a molecule, including bond distances and angles, and it provides accurate information about the spatial arrangement of the molecule relative to the neighboring molecules. These studies will have an impact in a number of areas, especially synthetic chemistry.

ID: MPS/DMR/BMAT(7623) 1206121 PI: Tirrell, David ORG: Caltech

Title: From Genes to Gels: Programming the Physical and Biological Properties of Multifunctional Protein Hydrogels

INTELLECTUAL MERIT: This project uses recombinant DNA technology to prepare multifunctional protein-based biomaterials. This approach to polymer synthesis provides near-absolute control over chain length and monomer sequence, and enables straightforward preparation of multifunctional materials. The physicochemical properties of protein polymers can be engineered further by exploiting methods for incorporating non-canonical amino acids into the protein chain. In this project, protein biosynthesis is harnessed to prepare new protein-based, multifunctional protein polymers that can be assembled into viscoelastic gels. The resulting materials are targeted for encapsulating and culturing pancreatic beta-cells and islets, and will contain functional domains thought to be important for beta-cell and islet survival. The bioactive regions of the proteins will be composed largely of elastin-derived sequences; these domains will alternate with leucine-zipper domains that permit assembly of the materials into physically crosslinked hydrogels through formation of coiled-coil aggregates. These gels will enable cell encapsulation through simple mixing of protein components under ambient conditions. The elastin sequences will be further tailored to contain the non-canonical amino acid azidohomoalanine, which enables control of viscoelastic properties through covalent crosslinking. This approach exploits the mild, bio-orthogonal character of the strain-promoted azide-alkyne cycloaddition to effect covalent crosslinking of protein gels. The goals of this work are (1) to engineer multidomain proteins that gel upon mixing, (2) to chemically crosslink protein hydrogels using mild, bio-orthogonal chemistries, and (3) to elucidate the behavior of cells encapsulated in physically and chemically crosslinked protein hydrogels.

BROADER IMPACTS: The research supported by this project will be integrated with a new high school science program, entitled "Genes to Gels," which introduces students to ideas and research at the interface between the biological sciences and materials science. The new program will bring to the Caltech campus a small number of students selected from Pasadena public high schools, for a three-week, summer hands-on laboratory experience. An important element of the program will be the commitment of the students' high school teachers to build on what the students do at Caltech, to develop demonstrations or experiments for use in their classrooms. Students will gain hands-on experience with each step of the process that takes us from "genes to gels." They will grow bacterial strains that carry artificial genes encoding leucine-zipper proteins, learn how to express and isolate proteins, and verify their molecular weights and purities by using gel electrophoresis and mass spectrometry. The proteins to be used in this work are well suited to experiments of this kind, because they can be purified easily by cycling through the lower critical solution temperature. With pure proteins in hand, students will prepare hydrogels and characterize the associated changes in viscosity by inversion tests, falling ball viscometry, and simple particle tracking. At the end of the program, all of the participants will work together to select and structure activities that can be productively transferred to the high school classroom. This program will impact a dozen classrooms and hundreds of high school students over the course of the project.

ID: MPS/DMR/BMAT(7623) 1237457 PI: Tirrell, David ORG: California Institute of Technology

Title: Important Areas for Future Biomaterials Investments

INTELLECTUAL MERIT: Biomaterials is a rapidly emerging component of the materials research community. Research in the area covers a broad range of activities that include the development of materials used for (1) delivery of therapeutic and diagnostic agents, (2) construction of medical devices that must be compatible with the living systems with which they are in contact, and (3) scaffolds for tissue engineering and regenerative medicine applications. Another aspect of the biomaterials field involves exploiting mimicry of, inspiration by, or co-opting of biological systems to enable creation of novel functional materials. For the purposes of the workshop, the various aspects of the field have been captured under the following topic headings: Cell-Material Interactions, Disperse Systems, Hard Materials and Composites, Soft Materials, and Thin Films and Interfaces. Expertise in all of these areas will be represented among the invited workshop participants. The workshop will involve a mix of plenary speakers and breakout sessions devoted to the topics just enumerated. A formal workshop report will be issued by the steering committee.

BROADER IMPACTS: The results of the workshop will be published in a report that will be widely disseminated and posted on a publicly accessible website. Additionally, a shortened version of the report, highlighting the most important results from the workshop, will be published in a leading journal. The steering committee also intends to return to NSF headquarters upon publication of the report to present their findings. The workshop report can be expected to guide the biomaterials community (educational institutions, commercial organizations, and funding agencies) in directing their resources toward the most critical and important biomaterials research challenges. For the relevant funding agencies, the workshop will help to identify their respective responsibilities in the biomaterials area and to foster robust and durable lines of communication among these agencies as they continue their ongoing investments. Workshop participants will include active researchers with diverse backgrounds in terms of topical area of expertise in biomaterials, career stage, institution, geography, gender, and ethnicity. These efforts will help ensure the conclusions of the workshop are representative of the biomaterials community, as a whole. Additionally, several graduate students will be invited to serve as assistants, allowing them the opportunity to observe the workshop and interact with prestigious members of the field. Finally, government agency representatives and selected members of industry will be welcome to attend the workshop.

DESCRIPTION (provided by applicant): Accommodating an aging world will pose significant economic and social challenges and will ultimately call for both paradigm-shift in our understanding, and biomedical interventions into the aging process. Extensive data demonstrate that when the systemic niches of organ stem cells are biochemically rejuvenated, stem cells endogenous to multiple old organs engage in productive tissue regeneration. Importantly, the old circulatory milieu rapidly and significantly inhibits the regenerative performance of endogenous stem cells in young organs. This proposal uses what is known about the role of the circulatory milieu in the rejuvenation of aged tissue repair, with the goal o determining the key molecular mechanisms that are responsible for the phenomena of heterochronic parabiosis. Until recently, it was technologically impossible to specifically label and interrogate the proteome of one animal in the setting of heterochronic parabiosis; however the development of cell-selective metabolic labeling of proteins with non-canonical amino acids via expression of mutant aminoacyl-tRNA synthetases in mammalian cells by the co-PI (David Tirrell) enabled this paradigm shifting approach. This high-risk high-reward project becomes feasible due to the united efforts, areas of expertise and experimental models of the Conboy and Tirrell research groups. The Conboy team has proficiency in studies of heterochronic parabiosis and characterization of the effects of defined factors on tissue regenerative capacity, whereas the Tirrell laboratory has pioneered and time-resolved and cell-selective proteomics. The paradigm changing outcomes of these studies are many fold: (1) revealing the youthful pro-regenerative and aged inhibitory proteomes of blood serum, (2) uncovering the mechanisms by which the circulation influences the regenerative performance of organ stem cells in muscle and brain/hippocampus; (3) generating a comprehensive data-base that is required for understanding the genetics of aging and importantly, (4) identifying novel ways to rejuvenate multiple organs and extend healthy life span via systemic administration of defined molecules that emulate the physiologically young blood circulation.

Non-Technical: This award by the Biomaterials program in the Division of Materials Research to California Institute of Technology is to explore new strategies for the design of tough, soft, protein-based materials. The materials of interest are hydrogels, polymeric materials that are highly swollen by water. Like the soft tissues of plants and animals, hydrogels exhibit a wide range of interesting and useful properties, including - most importantly - the capacity to respond to chemical and physical signals by changing shape and volume. But synthetic hydrogels are not very tough; in many cases it does not require much energy to cause them to fail. This project exploits the power of genetic engineering to design new protein-based hydrogels that show improved toughness. Artificial genes are used to encode protein sequences that dissipate energy upon deformation; the dissipated energy is then no longer available to cause the material to fail while in use. Improvements in toughness will lead to new applications of hydrogels in drug and gene delivery, tissue engineering, cell encapsulation and adhesion. The project provides important educational, training and outreach opportunities for graduate and undergraduate students. Additionally, the research activities of this project will be integrated with the "Genes to Gels," program at Caltech that will introduce high school students and teachers to research at the interface between the biological sciences and materials science. These high school students and teachers from the public schools in the greater Los Angeles area are expected to gain greatly with this hands-on research experience.

Technical: Hydrogels are crosslinked polymer networks of high water content. While their physical and chemical properties vary widely, most hydrogels form soft, elastic materials. This makes them excellent candidates to replace the natural extracellular matrix in soft tissue engineering and in vitro cell culture. But the same properties that make hydrogels appealing for these applications can also lead to brittle materials. This project uses genetic engineering to program the assembly of artificial protein hydrogels, with a specific focus on improving toughness. Genetic methods provide a level of control of chain sequence that is still not possible in conventional polymer synthesis, and the ease of sequence variation allows detailed exploration of the molecular processes that determine gel toughness. The toughening strategies developed in this work will establish general, broadly useful approaches to the design of tough, pliable materials. The project engages students at all levels from high school to graduate study, and includes science teachers drawn from high schools throughout the Los Angeles basin.

? DESCRIPTION (provided by applicant): We propose to develop methods to enable proteomic analysis of rare persister cells in phenotypically heterogeneous bacterial populations. Isogenic bacterial populations are characterized by phenotypic heterogeneity that includes variations in metabolic rates and responses to antibiotic treatment. Upon exposure to antibiotics, most bacterial cells die. But in many cases, a small subpopulation (usually < 0.1%) persists and, upon relief of antibiotic challenge, resumes growth. These persister cells have been observed for a wide variety of microbes treated with many types of antibiotics. The ability of pathogenic bacteria to persist and recover following antimicrobial therapy leads to chronic infections and the emergence of resistant strains. Understanding the mechanisms that enable persistence would constitute an important step toward treating and preventing chronic infections, but because studies of persister cells require analysis of small subpopulations of non-growing (or very slowly growing) cells, characterization of persisters has been difficult. We propose to develop and evaluate a general strategy for selective study of these cells at the proteomic level. Specifically we will use bio-orthogonal non-canonical amino acid tagging (BONCAT) and quantitative mass spectrometry to establish the time-dependent proteomic profiles of persister cells before, during, and upon recovery from antibiotic challenge. We aim to address the following questions: 1. How do persister cells respond to antibiotic challenge? 2. How do persister cells initiate growth following antibiotic challenge? and 3. What makes the persister cell subpopulation different from the antibiotic-susceptible majority? More generally, we will establish bioanalytical methods of broad utility in the study of bacterial persistence, chronic infection, and rare sub-populations in heterogeneous bacterial communities.

PROJECT SUMMARY/ABSTRACT We propose to explore the use of proline analogs to engineer and probe the biophysical behavior of insulin. Insulin contains a single proline residue ? at position 28 of the B-chain ? which is known to play critical roles in controlling the rates of onset of action and fibrillation of pharmaceutical preparations of the protein. Replacement of proline through conventional mutagenesis has led to FDA-approved rapid- acting insulins, but destabilizes the protein with respect to fibrillation. Conventional mutagenesis suffers from a fundamental limitation when applied to proline; any amino acid change converts the conformationally restricted cyclic proline residue to a more flexible acyclic one. We have recently found that replacement of the proline residue at position 28 of the insulin B-chain by (4S)-hydroxyproline ? through non-canonical amino acid mutagenesis ? yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. This approach allows one to alter critical molecular interactions around position B28 without sacrificing the unique conformational properties of proline. This proposal seeks to expand the known ?proline chemical space? that can be accessed in the bacterial expression of recombinant insulin. We will accomplish this objective by assessing the translational activity of a carefully chosen set of proline analogs in E. coli, by creating new prolyl-tRNA synthetases to activate the analogs of interest, and by analyzing the biophysical behavior of the resulting insulin variants by experimental and computational means. The proposed work will provide new forms of insulin with altered biophysical properties, expand the toolkit for engineering protein structure and function, and enhance our understanding of protein association and dynamics.