Lynmarie K. Thompson - US grants

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.

The aspartate receptor will be used as a model system for transmembrane signaling. It will be investigated using solid state nuclear magnetic resonance techniques and mutant receptor proteins to determine critical distances and geometric relationships in the combining site and accessibility of solvent and ligands to the combining site. Clustering of the receptor protein will also be studied. The results should yield a visualization at the molecular level of the receptor binding site and the role of receptor clustering and conformational changes in the receptor binding process. %%% Signal transduction is essential to biological organisms or organelles for the sensing of the environment, reacting to it if necessary and in intercellular communication. This research will focus on understanding how membrane receptors transmit information across cell membranes. The goal is to determine how structures of specific regions or active sites of the receptor molecules in the membrane change during the signaling process. Both the biophysical approaches used in this study and the insight gained into the molecular mechanisms of signaling should be generally useful for understanding more complex membrane receptors.

DESCRIPTION (Adapted from applicant's abstract): Transmembrane signaling is a fundamental step in a cell's ability to sense and respond to environmental signals, which is critical to processes ranging from bacterial chemotaxis to the coordinated function of cells in multicellular organisms. Ligand binding is thought to induce conformational changes or clustering to transmit the signal, but the mechanisms are ill-defined, in part because of the lack of structural information on the membrane-bound receptor proteins. The proposed research focuses on bacterial chemotaxis receptors (the related Asp and Ser receptors) as model systems for probing mechanisms of transmembrane signaling. The first goals are to map structural changes that occur in the periplasmic and transmembrane regions upon ligand binding--the initiation and propagation of the excitation signal. The approach is to integrate novel solid-state NMR techniques which measure selected distances with biochemical methods for targeting these measurements to the sites of interest. Site-specific isotopic labeling is achieved by using mutagenesis to introduce unique residues for subsequent labeling. Rotational resonance and REDOR are used to measure interhelical distances and how they change in response to ligand binding and methylation; additional measurements are designed to discriminate different types of motion. Application of a novel spin-diffusion technique also tests for piston motions of transmembrane helices. In the poorly understood cytoplasmic domain, distance measurements focus on testing coiled-coil models for the methylation region. Solution NMR will determine whether c-fragment dimers unfold during dissociation to monomers, to test the proposed inter to intramolecular coiled-coil model. Complementary solid-state NMR experiments probing receptor dynamics will identify dynamic regions and any changes in dynamics upon ligand binding or methylation. These experiments will directly measure proposed differences in structure and dynamics between signaling states. The proposed research provides a unique approach to obtaining direct information on molecular mechanisms of transmembrane signaling. In addition, the integrated biochemistry/NMR strategies developed for this study will be applicable to other important questions of membrane protein structure and function.

9908399

Abstract

This project involves the upgrade of a 500 MHz NMR spectrometer to provide state-of-the-art capabilities in order to support the research of a group of users in the Department of Chemistry at University of Massachusetts, Amherst. The major research projects will be facilitated by the availability of the new instrumentation. This research will include determination of the structure of the peptide-binding domains of the molecular chaperones DnaK and BiP; determination of bound conformation of signal sequences upon interaction with their receptors; determination of the structure of an SRP-binding RNA fragment alone and in complex with a polypeptide; studies of the mechanism of folding of the predominantly beta-sheet protein, cellular retinoic acid biding protein; structural analysis of myoglobin mutants as models for guanylate cyclase; conformational studies of model helical peptides; determination of the active site geometry of metalloproteins; determination of the structure and dynamics of membrane proteins, including E. coli chemotactic receptors and colicin A; studies of domains of the E. Coli chemotactic receptor that are involved in adaptation; and determination of the structures of ribosomal proteins and the RNAs to which they bind. Additional projects will further utilize the remaining time on this instrument.

DESCRIPTION (provided by applicant): In this proposal, we request a second renewal of our Chemistry-Biology Interface (CBI) Predoctoral Training Grant. In 1995 we launched a new interdisciplinary training program at the University of Massachusetts that built on existing strengths and formalized the faculty's commitment to collaboration between the physical and life sciences. We implemented a CBI curriculum that meshed with the requirements of the three participating graduate programs, Chemistry, Molecular &Cellular Biology, and Polymer Science &Engineering, yet enabled students with either chemical or biological backgrounds to supplement training in their home discipline with training in the complementary discipline. During the current funding period, our CBI Training Program has grown substantially: the number of participating departments has grown from 3 to 4, the number of CBI Training Faculty has grown from 16 to 19 and most notably, the number of formally affiliated students has increased from 14 to 61. The addition of Chemical Engineering builds our strengths in quantitative training and in biomaterials, and adds new opportunities in bioengineering. We anticipate continued growth of our CBI Program as additional departments, notably Physics and Computer Science, move into CBI-related areas. The CBI Program currently provides NIH support for six predoctoral students, and a University match supports one additional graduate student working at the chemistry-biology interface. To continue to foster the growing strength of the CBI program as it expands into new disciplines and trains increasing numbers of students, we request a modest increase in the number of NIH-funded Traineeships in years 3-5 of the of the grant period. The interdisciplinary training we provide is exemplified by the development of a new "Drug Design" course that is now an integral part of the CBI curriculum. In this popular course, students are provided with background lectures in chemistry and biology to bring them to a common ground, followed by weekly seminars by speakers from the pharmaceutical industry to introduce students to the full range of topics in chemistry and biology that are critical for successful design and development of new drugs. With this type of training in the methods and intellectual framework of both chemistry and biology, our CBI graduates are equipped to pose and solve significant biomedical questions in their future work.

DESCRIPTION (provided by applicant): The long-term goal of this project is to significantly increase the number of students from underrepresented groups who obtain Ph.D. degrees in biomedical fields. To this end, we will establish a one-year PREP internship that includes independent research and individualized programs of study, each with an interdisciplinary focus designed to increase the competitiveness of the PREP students for admission to rigorous biomedical graduate programs. Our project will capitalize on unique strengths of the University of Massachusetts Amherst (UMA): A) UMA leads the Northeast Alliance for Graduate Education and the Professoriate (NEAGEP), a highly interactive network of 15 minority-serving and research-extensive universities with demonstrated success in recruiting and retaining minority students STEM Ph.D. programs;B) UMA has well developed programs of interdisciplinary research and graduate education in biomedicine;C) UMA has developed effective multi-level mentoring programs to support diversity at all levels of academia. We will use these strengths to accomplish the following specific aims: 1) Use existing connections in NEAGEP and at UMA to recruit talented students from underrepresented groups;2) Pre-screen students through a well established eight-week summer program for undergraduate research (SPUR);3) Train near-peer and faculty mentors through an eight-week workshop series, "Entering Mentoring", focused on working with students from diverse backgrounds;4) Work with PREP participants and their mentors to formulate individualized programs of study based on interests, strengths and needs of students identified during SPUR;5) Start mentoring on first contact and continue through graduate school and beyond. During the program, students will rotate through two laboratories and have two sets of faculty and near-peer graduate student mentors. The Institute for Cellular Engineering, Chemistry-Biology Interface and Neuroendocrinology graduate training programs will provide an interdisciplinary focus through specific enrichment courses. Students will participate in regular social and professional development activities, many with NEAGEP doctoral students so that PREP participants will be part of a larger community of minority scholars and role models. Group PREP courses will focus on working in interdisciplinary and diverse groups in biomedicine, developing critical thinking skills and gaining an understanding of the responsible conduct of research. Faculty mentors, PIs and the Office of Graduate Recruitment and Retention will teach students how to apply to graduate school and to obtain pre- doctoral fellowships. At the end of the program, we anticipate that students will enroll in one of the numerous biomedicine graduate programs served by the participating interdisciplinary training programs or in graduate programs in other NEAGEP institutions. This program will help meet the growing need for biomedical researchers who can address issues of all segments of our population. The continuous multi-tiered mentoring and emphasis on interdisciplinary research inherent in the UMA PREP will increase the success of our students in doctoral programs and preparedness for biomedical research careers where the prevalence of multi-investigator projects is increasing. We expect our strategies to significantly increase the number of biomedical researchers from underrepresented groups. The strategies we devise will be transferrable and will help develop the diverse workforce needed to address disparities in healthcare and ensure continued international preeminence in biomedicine and biotechnology.

DESCRIPTION (provided by applicant): The chemotactic signal transduction pathway of Escherichia coli is representative of a large family of signal transduction pathways that are distributed throughout the Bacteria and Archaea. These pathways are already known to mediate chemotaxis and phototaxis in free-swimming and surface-associated bacteria, but are being recognized increasingly to regulate multicellular aggregation, pilus expression, and biofilm formation - medically relevant phenomena that are characteristic of pathogenic microbes. The relative simplicity of the E. coli system, combined with the well-developed and sophisticated tools for its study, provides a strong rationale to determine in detail the sensory physiology, biochemistry and structural biology of the E. coli pathway. Transmembrane signaling in the E. coli system occurs in the context of a heterogeneous cluster, or array, of receptor proteins that are associated with the cytoplasmic adaptor protein, CheW, and the central signaling kinase, CheA. Attractant binding to the receptors causes subtle conformational changes in the receptor that are somehow propagated across the membrane to produce two effects (i) inhibition of CheA, and (ii) stimulation of receptor methylation. Kinase inhibition exhibits a positive cooperativity indicative of regulation by a cluster of receptors. The key to understanding the detailed mechanism lies in understanding the manner in which these clusters of receptor complexes are assembled and remodeled during function. In this project we will investigate the interactions among receptors and with the signaling proteins in samples of successively greater complexity (in vitro to in vivo), to determine what structures and interactions are critical to the control of kinase and methylation activity. The simplest system, active complexes of the cytoplasmic domain, CheA, and CheW assembled on vesicle surfaces, will be studied in the greatest detail, with a combination of biochemical and biophysical tools (activity assays, disulfide crosslinking, fluorescence, and solid-state NMR). Studies of assemblies of complexes of the intact receptor in vesicles will determine how the interactions in the array are modulated by the other receptor domains and by ligand. Finally, in vivo fluorescence studies will investigate remodeling of receptor complexes and arrays during function. The approaches developed and insights gained will be applicable to other systems in which changes in both conformation and subunit association play a role in the mechanism of transmembrane signaling. PUBLIC HEALTH RELEVANCE: This project seeks to determine the function of signaling proteins in the chemotaxis pathway of E. coli through the use of isolated protein components reassembled into functioning signaling complexes and living bacterial cells. These samples will be tested with a combination of biochemical and biophysical techniques to correlate structure with activity. This E. coli system is representative of a large family of signaling pathways in microbes, including disease-causing bacteria, and therefore, the information obtained through the course of this project will be relevant to a great number of pathways, which may benefit the development of novel antimicrobial agents.

? DESCRIPTION (provided by applicant): This application requests the fourth renewal of our Chemistry-Biology Interface (CBI) Training Grant. In 1995 we launched a new interdisciplinary training program at the University of Massachusetts that built on existing strengths and harnessed our faculty commitment to collaboration between the physical and life sciences. We successfully implemented a curriculum that complemented the requirements of the participating graduate programs, adding enough training elements to efficiently train students with either chemical or biological backgrounds in the complementary discipline. The subsequent period (2000-2005) was one of tremendous growth: the number of CBI Program Members grew from 14 to over 60, as students well beyond those with CBI funding recognized the value of the training and community. Since then the program has continued to thrive, with membership remaining strong at 66 in 2010 and 72 CBI Program Members today. The success of the CBI program in establishing a collaborative community and interdisciplinary curriculum has led to major expansion of this research area on campus. In the current funding period we have seen significant investment by the university and the state in infrastructure and equipment, and the creation of a new Institute for Applied Life Sciences that connects many of the strong CBI research clusters with other life science researchers and engineers across campus. The 27 CBI Training Faculty continue to provide cross- training to over 70 students from the four participating graduate programs of Chemistry, Molecular & Cellular Biology, Chemical Engineering, and Polymer Science & Engineering. The CBI Program currently provides NIH support for 7 pre-doctoral students (reduced from 8 approved slots by NIH budget constraints), and a University match supports 2 additional Traineeships. Our inclusive approach combined with this university match has expanded training benefits 10-fold (7 NIH traineeships seed CBI training of 72 CBI Members). In view of the recent expansion of high TGE CBI Program Members from 35 in 2010 to 52 in 2015, we request an increase to 9 NIH-funded Traineeships. Central features of the CBI Program include the well-attended community-building Chalk Talk series, the popular Drug Design course featuring speakers from pharmaceutical companies, and an annual Retreat that forges connections with researchers at UMass Medical School. New training initiatives for the next funding period include a required Reproducibility in Research module, an optional computational drug discovery workshop and course, a CBI Toolbox Tour element of Chalk Talk to stimulate training and discussion of our significantly expanded instrumentation capabilities, and an increase to four CBI student-invited seminars to build even stronger community ties to all four participating programs. Thus the CBI curriculum and community are rich in opportunities for training in the methods and intellectual framework of both chemistry and biology, which prepares our CBI graduates to pose and solve significant biomedical questions in their future work.

Abstract This application requests the fourth renewal of our Chemistry-Biology Interface (CBI) Training Grant. In 1995 we launched a new interdisciplinary training program at the University of Massachusetts that built on existing strengths and harnessed our faculty commitment to collaboration between the physical and life sciences. We successfully implemented a curriculum that complemented the requirements of the participating graduate programs, adding enough training elements to efficiently train students with either chemical or biological backgrounds in the complementary discipline. The subsequent period (2000-2005) was one of tremendous growth: the number of CBI Program Members grew from 14 to over 60, as students well beyond those with CBI funding recognized the value of the training and community. Since then the program has continued to thrive, with membership remaining strong at 66 in 2010 and 72 CBI Program Members today. The success of the CBI program in establishing a collaborative community and interdisciplinary curriculum has led to major expansion of this research area on campus. In the current funding period we have seen significant investment by the university and the state in infrastructure and equipment, and the creation of a new Institute for Applied Life Sciences that connects many of the strong CBI research clusters with other life science researchers and engineers across campus. The 27 CBI Training Faculty continue to provide cross- training to over 70 students from the four participating graduate programs of Chemistry, Molecular & Cellular Biology, Chemical Engineering, and Polymer Science & Engineering. The CBI Program currently provides NIH support for 7 pre-doctoral students (reduced from 8 approved slots by NIH budget constraints), and a University match supports 2 additional Traineeships. Our inclusive approach combined with this university match has expanded training benefits 10-fold (7 NIH traineeships seed CBI training of 72 CBI Members). In view of the recent expansion of high TGE CBI Program Members from 35 in 2010 to 52 in 2015, we request an increase to 9 NIH-funded Traineeships. Central features of the CBI Program include the well-attended community-building Chalk Talk series, the popular Drug Design course featuring speakers from pharmaceutical companies, and an annual Retreat that forges connections with researchers at UMass Medical School. New training initiatives for the next funding period include a required Reproducibility in Research module, an optional computational drug discovery workshop and course, a CBI Toolbox Tour element of Chalk Talk to stimulate training and discussion of our significantly expanded instrumentation capabilities, and an increase to four CBI student-invited seminars to build even stronger community ties to all four participating programs. Thus the CBI curriculum and community are rich in opportunities for training in the methods and intellectual framework of both chemistry and biology, which prepares our CBI graduates to pose and solve significant biomedical questions in their future work.

FASEB Science Research Conference on `Molecular Biophysics of Membranes' Summary Support is requested for a FASEB Science Research Conference on Molecular Biophysics of Membranes. The 2018 conference will be the 15th in a highly successful 30-year series of FASEB conferences devoted to this area of research. Membrane structural biology and membrane biophysics is a rapidly advancing field. Over 50% of all drug targets, prokaryotic or eukaryotic, are membrane proteins, but they are difficult targets due to numerous challenges. Technical developments in expression, purification, and crystallization of membrane proteins have fueled an exponential increase in the number of membrane protein structures that are solved annually. Developments in cryo-electron and super-resolution microscopy have further resolved architectures of molecular machines that work in and on membranes of viruses, bacteria, and eukaryotic cells. These and many other contemporary topics will be covered in a 5-day meeting with 9 scientific sessions and 2 keynote lectures. The 2018 summer conference includes a strong focus on membrane proteins of bacterial and other pathogens. It will bring together ~ 110 junior and senior scientists in a collegial atmosphere in Olean, NY, with 37 of them giving invited talks and an additional 16 selected to give short talks based on their submitted abstracts. All those not selected for short talks will have the opportunity to advertise their posters in single-slide ?poster preview? talks. This and daily ?meet the experts? sessions will promote networking between researchers of all career stages. The meeting has a long tradition of bringing together biophysicists, biochemists, virologists, microbiologists, cell biologists, and physiologists in an intimate setting to share the latest developments in their fields. Biophysicists and other basic scientists are exposed to interesting applied biological problems, and translational biologists learn about new ideas and techniques for studying and understanding molecular processes that happen in and around membranes. The requested funds will be used to support the registration and travel expenses of a diverse set of speakers.