Valerie Copie - US grants

06/19/98 This proposal will fund the purchase of a computing cluster to support computational biology research at Montana State University. The computing equipment will be used for multidisciplinary research in the general areas of Neurosciences, Population Ecology, Biochemistry, and Structural Biology. The major users will be 12 faculty from four different departments at Montana State University: Biology, Chemistry and Biochemistry, Mathematics, and Computer Science. A total of 11 postdocs and 27 graduate students in these faculty labs will use the equipment for their research projects. This computer cluster will also directly support research projects involving numerous undergraduate students, including those enrolled in several special programs for minority students. These students will have access to the equipment though their involvement in research projects sponsored by the faculty users.

Funds are requested for the acquisition of a liquid state 14.1 Tesla (600 MHz proton frequency) NMR spectrometer to be used by a diverse group of highly productive scientists from the Departments of Chemistry and Biochemistry, Microbiology, Veterinary Molecular Biology, and Plant Pathology at Montana State University, as well as Montana Tech, and the University of Montana. This instrument would expand the NMR-based research capabilities of a large group of MSU investigators currently involved in structural biology research. The majority of the projects require the collection of high resolution NMR data on large proteins, or on peptide-protein complexes, in solution. Acquisition of a 600 MHz NMR spectrometer would also free up NMR time on the current 500 MHz NMR spectrometer, making it more available to chemists involved in the synthesis and characterization of bioactive compounds. At present, the MSU 500:MHz NMR spectrometer operates 24 hours/day, and is used 99% of the time by members of the membrane protein research group, and by Dr. Copie's research which concerns the structure-function study of proteins, using recently developed multinuclear and multi-dimensional solution NMR methods. The growing demand for 500 MHz NMR time already exceeds the amount of instrument time available. Acquisition of a 600 MHz spectrometer would accomplish two goals: 1) It would enable members of the structural biology group to take advantage of the greater sensitivity and resolution of a 600 MHz NMR instrument to advance their structure-function studies on proteins, or on peptide- protein complexes, and 2). It would provide easier access to the 500 MHz NMR spectrometer for a wide range of organic chemistry research. With the new instrument, the investigators listed below, along with a group of several secondary investigators, will be in a position to expand their research to larger molecules, and to carry out competitive research related to structure and function in biological systems. The availability of the 14.1 T spectrometer will allow researchers to access the currently overbooked 11.7 T instrument.

Copie
9984562


This research program focuses on solving the three-dimensional structure of the intracellular domain of a naturally occurring neurotrophin receptor isoform, ICD-TK- trkC, which was recently shown, together with P75 receptor activation, to be effective at promoting neuronal differentiation. Modern multi-dimensional (1H, 15N, 13C) NMR methods will be used to determine its 3D structure in solution at high resolution. The goal of this work is to provide structural and molecular data that, together with site-directed mutagenesis experiments and biochemical assays, will bring insight as to the mechanism(s) by which TK- trkC promotes differentiation of neural cells.

The objective of this research is to provide fundamental knowledge about key biochemical interactions regulating neuronal development. This research will integrate the PI's major educational initiatives, including the recruitment of Native American students into the sciences, establishment of "hands on" experiences and "mini-rotations" in research laboratories for undergraduate students, and the organization of seminar series and mentorship programs to encourage under-represented student groups to pursue majors and/or research project in science. This project is an integral component of the PI's involvement with the cross-disciplinary research and educational initiatives being developed at Montana State University through an NSF-sponsored (IGERT) graduate training program.

This award provides support for purchase of equipment to be used to establish a multi-user, integrated core equipment facility for cellular and molecular biological research. The equipment includes centrifuges, a microinjection apparatus, a spectrophotomer and an inverted microscope. The facility will be shared by faculty in two departments: a newly formed Department of Cell Biology and Neuroscience and an existing Department of Chemistry and Biochemistry. A shared focus of all these investigators is the elucidation of the cellular and molecular mechanisms underlying neural development and/or nerve regeneration. All are now physically located in adjacent labs, and given the overlap in their research methodologies, will be able to share the equipment supported through this award.. Some of the major equipment items requested would replace existing items that are close to 25 years old and in serious disrepair, while others will provide new or expanded capabilities. All major users are members of the NSF sponsored IGERT doctoral training program at the University. This program in Complex Biological Systems is designed to train graduate students to approach biological problems from a multitude of levels, from the molecular to the systems level, using a variety of approaches drawn from cellular and molecular biology, structural biology, computational biology and systems neuroscience. Thus the IGERT trainees will benefit considerably from access to the equipment which will be used in both course work and in thesis research. The equipment will also be available for use by a number of undergraduate students who undertake independent research projects as part of their education.

The long-term goal of this project is to provide a better understanding of the interconnections between internal dynamics, protein structures, and functions. The internal dynamics of backbone and sidechain atoms of wild-type and functionally altered mutants of the 25 kDa tryptophan repressor (TrpR) protein will be studied using NMR relaxation approaches. TrpR is part of a large family of bacterial proteins that regulate the expression of metabolic genes by binding to DNA operator sequences in response to cellular needs. The repressor's affinity for DNA is controlled by L-tryptophan (L-trp), an allosteric effector that activates the TrpR. The intrinsic flexibility of the TrpR protein structure is thought to be at the origin of the non-cooperativity of binding of the L-tryptophan corepressor to TrpR, and of the non-local long-range effects observed in a temperature-sensitive mutant of the tryptophan repressor protein, L75F-TrpR, which cannot be simply explained by small structural changes when compared to the wild-type TrpR. A second TrpR mutant of interest is A77V which, like L75F, is structurally similar to wild type TrpR and possesses biophysical features analogous to those of L75F-TrpR. Despite similar structures and biophysical features, the two TrpR mutants yield very distinct phenotypes and have very different L-trp cofactor binding properties. Such biochemical differences cannot be explained by the presence of distinct structural changes. The hypothesis to be examined with the NMR relaxation experiments is that the source of the differential L-trp binding properties of the A77V and L775F TrpR proteins originate from differences in intrinsic flexibility and molecular mobility which cannot be identified from inspection of the protein structures. The objective of this project is to provide new insights into the biochemical role of internal motions in modulating TrpR-L-trp-cofactor complex formation and TrpR-DNA recognition.

This research will train graduate and undergraduate students in cross-disciplinary research involving NMR-based structural biology and fundamentals of protein chemistry. In addition, the PI will use knowledge obtained from this research to develop a graduate level course in biophysics focusing on the thermodynamics of ligand binding, protein-protein and protein-nucleic acid interactions, role of structural stability and dynamics in modulating protein functions, and allosteric regulations of multivalent protein systems.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Crenarchaeal viruses such as SSV1 and STIV inhabit extreme thermal and acidic environments (T> 80oC; 4.0 > pH > 1.0) such as those found in Yellowstone National Park. SSV1 and STIV are among the best characterized of these viruses, and thus serve as important model systems for study. Sequence analyses of the SSV1 and STIV genomes, and those of other viruses from extreme thermal environments, are unable to suggest functions for most of the encoded proteins. The proposed research focuses on the use of structural biology to investigate the functional roles of the SSV and STIV proteomes, and on interactions in host organism Sulfolobus solfataricus. Structures of viral and host proteins of unknown function will be determined using crystallographic and NMR techniques. In many cases, these studies are expected to reveal structural similarity to proteins of known function, or to otherwise suggest functions that in collaboration with Dr. Mark Young, Montana State University, can be tested using biochemical and genetic experiments. Together, these studies will contribute to an increased understanding of the SSV and STIV life cycles, and the molecular interplay between these viruses and their crenarchaeal hosts.

Broader Impact: Much of the ongoing work within the MSU Thermal Biology Institute (TBI) including the characterization of hyperthermophilic viruses is being communicated to the general public through outreach programs. Target groups include K-12, tribal colleges, and other undergraduate institutions. In addition, TBI partners with the National Park Service /Yellowstone National Park in outreach to the general public within the Park. There is strong integration of the proposed research with the educational goals of the PI, Co-PI, the Department, the University, and the NSF. Students at the high school, undergraduate, doctoral and post-doctoral levels will receive significant training in biochemistry, structural biology and thermal biology. The involvement of under represented groups, particularly women and Native Americans, is actively encouraged.

DESCRIPTION (provided by applicant): Support is requested to acquire a high-sensitivity 600 MHz triple resonance (1H, 13C, 15N) TCI cryoprobe and to upgrade an existing 600 MHz (1H Larmor frequency) NMR spectrometer console at Montana State University (MSU) to a more automated and high-throughput data collection platform (Bruker Avance IIITM). This instrumentation will be used by a diverse group of researchers from MSU Departments of Chemistry and Biochemistry, Veterinary Molecular Biology, Chemical and Biochemical Engineering, the Center for Biofilm Engineering, the Department of Cell Biology and Neuroscience, and the NIH Rocky Mountain Laboratory. Currently, none of the solution NMR spectrometers in the MSU Structural Biology and Chemistry NMR Facility are equipped with a cryoprobe, and the requested instrumentation and upgrade of the spectrometer console hardware will be the first of its kind on campus. The requested instrumentation upgrades will enhance sensitivity and maximize throughput of MSU 600 MHz solution NMR spectrometer. It will contribute significantly to the successful completion of challenging NMR-based structural biology projects and the expansion of NMR metabolomics research programs that are being developed on campus. The very substantially enhanced sensitivity achieved with the requested instrumentation will allow acquisition of experiments and studies on challenging biological samples, which are currently not possible because of limited sample solubility, limited sample stability, or limited instrument time availability. The console upgrade will allow automated shimming and performance optimization, and will provide much more efficient use of the recently purchased automatic sample loading system (Bruker SampleJetTM). The combination of the console upgrade and the automatic sample loading system will allow more effective 24/7 utilization of the 600 MHz NMR instrument. The cryoprobe and upgraded console will greatly facilitate the expansion of our existing NMR metabolomics capabilities (which include ChenomxTM and AMIXTM software for metabolite profiling analysis). Enhancement of NMR structural biology and metabolomics infrastructure is desired by a number of prospective users and supports the development of a new NIH-funded P20 Center for the Analysis of Cellular Mechanisms and Systems Biology. MSU's DRX 600 NMR instrument is used quite heavily, but the enhanced sensitivity and performance provided by the requested upgrades will allow this instrument to accomplish much more. The addition of the requested instrumentation will leverage significantly enhanced biomedical research capabilities for a broad group of researchers on the MSU-Bozeman campus. The requested instrumentation will also provide opportunities for new research directions in translational biomedical research, will support expanded systems biology approaches at MSU, and will contribute to the training and employment of numerous graduate students, post-doctoral fellows, and research staff. PUBLIC HEALTH RELEVANCE: We request support from the NIH to enhance the nuclear magnetic resonance (NMR) biomedical research capabilities of Montana State University. The requested instrumentation will enhance the research capabilities of a broad group of scientists involved in better understanding the molecular basis of iron homeostasis, host-pathogen interactions, viral infections, Alzheimer's, diabetes, AIDS, and other important human diseases.

An award is made to Montana State University to purchase a Bruker AVANCE III 500 MHz NMR spectrometer equipped with a sensitivity-enhanced Prodigy TM broadband cryoprobe. The new instrument will enhance research and education at all levels. It will strongly benefit student education at both the graduate and the undergraduate level by enabling student training in new, powerful small molecule NMR analytical techniques. MSU provides direct, hands-on training to students in state-of-the-art instrumentation, which is taught in venues ranging from summer undergraduate research programs to sophomore organic laboratory courses to graduate level instructional programs. The new AVANCE III 500 NMR with its enhanced capabilities will also enable them to enhance participation of minority Native American students through remote instrument access that will provide NMR spectra to students enrolled in sciences classes at remote Montana Tribal Colleges and at other small colleges across the state.

The cryoprobe-equipped AVANCE III 500 NMR will allow researchers to use NMR for chemical structure elucidation and the global analysis of small molecule metabolites (e.g. metabolomics) research, will enhance usage by permitting higher throughput data collection, and will facilitate studies in applications with inherently low selectivity. In addition, the new instrument will be the workhorse NMR spectrometer for the university and will greatly expand Montana State University's NMR research capabilities for acquisition of multidimensional (1D, 2D, 3D/nD) heteronuclear (1H, 13C, 15N, 31P) NMR spectra of small molecules. The new instrumentation, which will replace MSU's aged 500 NMR spectrometer, will boost research productivity and student education. The new instrument is absolutely essential to maintain the university's research productivity, educational quality, and outreach mission, and for successful recruitment of new faculty.

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding a collaborative local team at Montana State University comprised of Dr. Eric Shepard (PI), Dr. Joan Broderick, Dr. Valérie Copié, and Dr. Robert Szilagyi to investigate the minimal protein structural requirements for generating and controlling organic radical chemistry in biology. The largest known superfamily of enzymes in Nature is the radical S-adenosylmethionine (SAM) superfamily. These metalloenzymes contain an iron-sulfur cluster that is responsible for the coordination and cleavage of the essential cofactor SAM. The cleavage of SAM generates a radical intermediate species, known as the 5'-deoxyadenosyl radical, which is responsible for propagating a diverse array of chemical transformations essential to life. Examples of such transformations include DNA repair, RNA modification, protein activation, vitamin biosynthesis, and cofactor formation. This project employs parallel synthetic, spectroscopic, and computational investigations to develop and characterize iron-sulfur cluster complexes that are functional in SAM chemistry. These synthetic compounds are used to understand the specific SAM-based carbon-sulfur bond activation event that leads to 5'-deoxyadenosyl radical formation. Students are being trained in interdisciplinary research that encompasses biochemical, spectroscopic, kinetic, computational and synthetic methodologies. Outreach and educational activities are within the Native American and home-schooling communities from rural Montana. As part of these activities young scientists are being exposed to basic chemistry/biochemistry laboratory exercises, as well as advanced scientific problem solving techniques.

Enzymes in the radical S-adenosylmethionine (SAM) superfamily catalyze an incredible array of chemical transformations essential for life's processes. This diversity is propagated through a stoichiometrically simple Hydrogen-atom abstraction event. At the core of radical SAM catalytic processes is a redox active [4Fe-4S] cluster that harbors a site-differentiated iron site that promotes the bidentate coordination of SAM. Using a retrosynthetic approach, minimalist protein maquettes that coordinate site-differentiated [4Fe-4S] clusters are prepared to probe the importance of protein environment in fine-tuning the stability, reactivity, and selectivity of SAM based radical reactions. Complementary NMR, EPR, CD, UV-vis, HPLC, SAXS, XAS, and Mössbauer techniques, along with multi-layered DFT/MO/MM computational models are techniques that interrogate the structure, stability, and reactivity of the maquette complexes. Chemical, structural, and mechanistic characteristics of the maquette complexes are investigated through structure-function relationships. The insights gained from [4Fe-4S] maquette studies may contribute to understanding how and why Nature uses this platform as its preferred method for generating and propagating radical reactions.

What factors allow single-celled microbial organisms to not only survive, but actively seek out environments that humans consider highly toxic? Scientists have learned much about how these tiny living creatures function, facilitating breakthroughs in medical, energy, and bioremediation sciences. However, the understanding of how microorganisms grow and thrive in harsh environments remains largely cloaked in mystery. The ability to predict how microorganisms will respond to environmental changes or the knowledge of the types of reactions that occur inside and outside their cells is vastly under-represented. In a large part, this is due to the limited ability to identify the small molecules produced and consumed by microbes (metabolites). This unknown microbial metabolite landscape limits the understanding of the microbial processes playing key roles in the regulation of biogeochemical cycles, bioremediation, bioenergy production, as well as the human microbiome. In this work, state of the art techniques will be used to find and characterize metabolites that up until now have been largely invisible to researchers. The work will result in excellent training opportunities for undergraduate and graduate students, especially from under-represented groups such as Native Americans, as well as a series of lectures at the Thermal Biology Institute that provide an opportunity for effective outreach and give the public a view of the importance of microbes.

This project focuses on the discovery of unknown microbial metabolites produced by bacteria using the Gram-negative soil bacterium Agrobacterium tumefaciens strain 5A as model. This bacterium is a model for understanding how microbes metabolize arsenic, a critical environmental toxin found in contaminated soils and water supplies, and a top priority for bioremediation efforts. Focusing on how microbes metabolize arsenic is important because microorganisms influence arsenic toxicity and bioavailability in every environment thus far studied. Thus, characterization of arsenicals produced by microbes are a focal point of this project that will employ state-of-the-art nuclear magnetic resonance (NMR) and mass spectrometry (MS) metabolomics technology to identify unknown microbial compounds, including arsenicals (i.e. methylated species, arsenolipids, arsenosugars). Importantly, the analytical approaches developed in this project will be applicable to many facets of biology. The project will greatly enhance researchers' abilities to probe the richness of the metabolomes of microbes, and to gain a much better appreciation for the diversity of microbial unknown small molecules. Focusing on A. tumefaciens as a model system will enable the identification and structural characterization of novel organo-arsenic compounds and arsenolipids. Results from these studies will bring transformative knowledge to the field of microbe-arsenic interactions relevant to scientific research aimed at predicting how arsenic disrupts/alters bacterial metabolism in situ, with broader implications for biogeochemical carbon and nitrogen cycling in nature when microbes must deal with arsenic.

Project Summary The goal of this project is to determine whether metabolism and the gut microbiome underlie hallmark features of the neurodegenerative disease, Familial Dysautonomia (FD). While clinical hallmarks of FD involve the sensory and autonomic nervous system, including cardiovascular instability and orthostatic hypotension with bouts of hypertension, another cardinal feature is impaired gastrointestinal (GI) tract motility. The human GI tract is regulated by over 500 million intrinsic neurons, called the Enteric Nervous system (ENS). The ENS is a component of the Autonomic Nervous System and has been shown to be severely reduced in neuronal number in FD patients. Furthermore, FD patients and mouse models for FD are underweight and mice are essentially devoid of subcutaneous white adipose tissue. The underlying etiology for their reduced mass is not known but recent data has shown that mitochondrial function is impaired in FD patients and mice. The ?gut? and ?brain? communicate extensively and accumulating data demonstrate the strong role the gut microbiome exerts on both metabolism and the nervous system, resulting in exacerbation of neurodegenerative disorders. We hypothesize that FD patients and mice are underweight because they suffer from a global metabolic syndrome induced by a combination of gut microbiome alteration, impaired energy homeostasis and mitochondrial dysfunction, and reduced gut regulation by the enteric, autonomic and sensory nervous systems. Using a multi-disciplinary approach, we will analyze the gut microbiome and metabolome of FD patients and manipulate these systems in mouse models of FD to identify and sort causal mechanisms mediating both metabolic impairments and neuronal health. Although specifically focused on FD, our results will broadly apply to other neurodegenerative diseases, where metabolism and the microbiome are thought to play a role.

RESEARCH PLAN Project Summary of Funded Parent Grant 1R01DK117473-01A1 The goal of this project is to determine whether metabolism and the gut microbiome underlie hallmark features of the neurodegenerative disease, Familial Dysautonomia (FD). While clinical hallmarks of FD involve the sensory and autonomic nervous system, including cardiovascular instability and orthostatic hypotension with bouts of hypertension, another cardinal feature is impaired gastrointestinal (GI) tract motility. The human GI tract is regulated by over 500 million intrinsic neurons, called the Enteric Nervous system (ENS). The ENS is a component of the Autonomic Nervous System and has been shown to be severely reduced in neuronal number in FD patients. Furthermore, FD patients and mouse models for FD are underweight and mice are essentially devoid of subcutaneous white adipose tissue. The underlying etiology for their reduced mass is not known but recent data has shown that mitochondrial function is impaired in FD patients and mice. The ?gut? and ?brain? communicate extensively and accumulating data demonstrate the strong role the gut microbiome exerts on both metabolism and the nervous system, resulting in exacerbation of neurodegenerative disorders. We hypothesize that FD patients and mice are underweight because they suffer from a global metabolic syndrome induced by a combination of gut microbiome alteration, impaired energy homeostasis and mitochondrial dysfunction, and reduced gut regulation by the enteric, autonomic and sensory nervous systems. Using a multi-disciplinary approach, we will analyze the gut microbiome and metabolome of FD patients and manipulate these systems in mouse models of FD to identify and sort causal mechanisms mediating both metabolic impairments and neuronal health. Although specifically focused on FD, our results will broadly apply to other neurodegenerative diseases, where metabolism and the microbiome are thought to play a role.

This award is supported by the Major Research Instrumentation, and the Chemistry Research Instrumentation programs. Montana State University is acquiring a 400 MHz nuclear magnetic resonance (NMR) spectrometer equipped with an automatic sampler to support Professors Michael Mock, Valerie Copie, Matthew Cook, and other colleagues. This spectrometer allows research in a variety of fields such as those that accelerate chemical reactions of significant economic importance, as well as permitting study of biologically relevant species. In general, NMR spectroscopy is one of the most powerful tools available to chemists for the elucidation of the structure of molecules. It is used to identify unknown substances, to characterize specific arrangements of atoms within molecules, and to study the dynamics of interactions between molecules in solution or in the solid state. Access to state-of-the-art NMR spectrometers is essential to chemists who are carrying out frontier research. This instrument is an integral part of teaching as well as research and research training of students in chemistry and biochemistry at this institution and nearby Tribal Colleges as well as the University of Montana and other small colleges across the state.

The award of the NMR spectrometer is aimed at enhancing research and education at all levels. It especially facilitates studies of molecular catalysts for nitrogen reduction and ammonia oxidation of relevance to the production of fertilizers. The instrumentation is also used for monitoring cascade reactions and controlling site selectivity in cross-coupling reactions and activating inert hydrocarbons using early transition metals. In addition, it provides information to aid exploration of halogen bonding in supramolecular and self-assembly chemistry. The spectrometer is also used to study mechanisms in organic and organometallic chemistry.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.