David M. Dooley - US grants

9322037 Jennings Montana State Univ This award from the Inorganic, Bioinorganic, and Organometallic Program of the Chemistry Division will support an annual workshop in organometallic chemistry. The participants will be organometallic chemists from academia, industry, and private and government research organizations. The goals of the workshop are to facilitate communication among researchers in the different kinds of organizations and to foster the exchange of new ideas much earlier than is possible through formal publications. The format of the workshop is designed to encourage maximum participation by all attendees, and the plan for selecting attendees will ensure broadly based participation. The topics to be discussed will span a broad spectrum from synthesis of new organometallic compounds to the utilization of such compounds as catalysts or as precursors for making new materials. However, the topics of "Benign Chemical Synthesis and Processing" and "Critical Technologies" will form the overall themes of the workshop.

This award from the Chemistry Research Instrumentation and Facilities Program will help the Department of Chemistry at Montana State University upgrade its x-ray diffraction laboratory. Major projects supported by acquisition of this equipment will include: Organic Synthesis; Organometallic Transformations of Organic Systems; Preferred Structures of Non-Rigid Molecules; Rare Earth Doped Crystals for Laser Materials; Characterizations of Bioactive Metabolites from Plant Microbes; Polynues; Polynuclear Bridged Mixed Valence Platinum Compounds. Single crystal x-ray crystallography is the most powerful analytical method for structure determination of solids. In synthetic inorganic, organic, bioinorganic and organometallic chemistry, single crystal x-ray diffraction is an invaluable tool to characterize molecular structure. The information gained from the knowledge of the molecular composition and structure helps to develop new reactions of potentially general intereral interest in catalysis or synthesis.

This award from the Chemistry Research Instrumentation and Facilities Program will aid in the purchase of mass spectrometry data systems in the Chemistry Department at Montana State University. The research to be performed with this instrumentation include the following: Studies of various high pressure gas phase ionic processes; the identification of the neutral products formed in gas phase ionic processes; fundamental studies of desorption ionization processes associated with FAB and Electrospray mass spectrometry; the detection of environmentally interesting atmospheric components at the low- and sub- parts per trillion level; organic transformations facilitated by platinum(II) and other transition metals; biological control and signalling mechanisms involving macromolecular receptors in visual and neutrophil systems; the production of Taxol and Taxanes from microbiological sources; and the identification of lipid peroxidation products generated by environmental toxins. Mass Spectrometry (MS) is a technique used to probe intimate structural details and to obtain the molecular compositions of a vast array of organic, bioorganic and organometallic molecules. The computer data systems allow state-of-the-art control of research grade mass spectrometers which gives the chemist one of the most powerful tools available for the characterization of compounds. The acquisition of this capability in mass spectrometry is essential for the prosecution of frontier research in many fields of chemistry.

9317962 Teintze The Department of Chemistry and Biochemistry needs a new preparative ultracentrifuge which would be located in Gaines Hall. There is at present only one functional ultracentrifuge in the Department, a 19 year old L3 50 instrument belonging to Dr. Dratz, which is incapable of meeting the Department's present and future needs. This obsolete machine has been derated by the manufacturer (Beckman Instruments) and can no longer be used with many rotors, such as the SW60 Ti rotor, which Dr.Teintze brought with him and needs for his work. Furthermore, one ultracentrifuge cannot handle the increased use resulting from Dr. Teintze's arrival in the Department in the fall of 1992 and that of Dr. Dooley in June of 1993. Two additional investigators in the Department, Drs. Hapner and Jackson, also need ultracentrifuges for their research. The Department also lacks a large capacity fixed angle rotor. The Type 30 rotor that it owns has been derated to a maximum of 10,000rpm because of its age (21 years) and amount of use, which makes it useless for the required applications. We are therefore applying for matching funds to purchase a new ultracentrifuge and funds for a preparative rotor. Ultracentrifuges are essential equipment for all researchers in biochemistry and molecular biology. The new ultracentrifuge will be used primarily by the labs of Drs. Teintze, Dratz and Dooley, so the proposal contains descriptions of their projects that require its use. These projects are all funded by ongoing NIH or NSF research grants. The instrument will be used for density gradient analysis of proteoliposomes, membrane puri fication, cell fractionation, and DNA purification. Project 1, entitled "The Mechanism of Protein Insertion into Lipid Bilayers", requires the most centrifugation time. It involves isolating the DNA coding for a bacterial membrane protein, making site specific mutations in the gene, and determining the effect of the resulting amino acid changes on the ability of the protein to insert into a membrane, both in the bacterium and in vitro using purified protein and phospholipid vesicles. The membrane protein and DNA purifications use ultracentrifugation, and the analysis for insertion in to lipid vesicles requires it also. This is done using density gradients that take 18 hours using the SW60 Ti rotor, but require centrifugation for 2 or 3 days using the rotors that can be used in the old L3 50 centrifuge. These long runs tie up the centrifuge for long periods of time, interfering with their use for preparative work for this and other projects. Project 2, which involves purification of the human membrane CFTR and its insertion into phospholipid vesicles for the purpose of developing a therapy for cystic fibrosis, also utilizes similar techniques requiring the new ultracentrifuge. Drs. Dratz and Dooley (Projects 3 and 4, respectively) would also be primary users of the requested instrument for membrane and protein purification. Project 3 involves isolation of rod outer segment membranes and rhodopsin; project 4 requires the isolation and purification of a number of bacterial membrane enzymes. All of these procedures would require ultracentrifugation using a preparative rotor such as the Type 45Ti we are requesting funds to purchase. r r * - 2 J 9 : J ; O ; G j X Z G _ ` k _ u WV F ^ F ;F t + F ~ u j V W v j j j j j9317962 Teintze The Department of Chemistry and Biochemistry needs a new preparative ultra centrifuge which would be located in r <> # % p r ! ! ! ! ! ! F " r r 3 Times New Roman Symbol & Arial 1 Courier " h 0 E0 E = abstract Deseree King, BIR Deseree King, BIR

catherine p. thomas

9723715 Dooley The biochemistry, biosynthesis, structures, reactivity, and function of nitrous oxide reductase, the terminal enzyme in bacterial denitrification, is being investigated. Denitrification is a key component of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The ecology involving nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N20 to the atmosphere, where it may contribute to ozone depletion and global warming. This research addresses several outstanding issues in the biochemistry of denitrification, in the bacterium Achromobacter cycloclastes, with the goal of a thorough molecular understanding of this pathway. The roles of the proteins encoded by the genes nosD, F,Y,L,X, which are (or may be) required for the biosynthesis of nitrous oxide reductase, are being examined. The general strategy is to clone and over-express the soluble, periplasmic nos-coded proteins, which may be involved in the biosynthesis of the catalytic site of nitrous oxide reductase, thereby insuring sufficient material for detailed biophysical and functional studies. Site-directed mutagenesis, together with variable -temperature absorption, CD, MCD, EPR and resonance Raman spectroscopy will be used to probe the nature of the catalytic site in nitrous oxide reductase (thought to be the Cuz site), and the mechanism of reduction of nitrous oxide, including the interactions with physiological electron donors. Concerted efforts are underway to develop rapid, generally applicable schemes to purify nitrous oxide reductase, with the objective of obtaining crystals of nitrous oxide reductase suitable for x-ray diffraction analysis. Results of these experiments should be applicable to major themes in modern biochemistry such as metalloenzyme structure and function; metal ion metabolism and the biosynthesis of metalloproteins; and the recognition and acti vation of kinetically-inert small molecules. This research project seeks to understand how bacteria in soil and water convert nitrate, an important source of nitrogen for plants, into nitrogen gas, which is released to the atmosphere. A substantial fraction of the fertilizers applied to crops are wasted as a result of this conversion. The organisms responsible contain an enzyme known as nitrous oxide reductase, which plays a critical role in the overall process. A major goal of the project is to understand how this enzyme is made, its structure, and exactly how it functions. Understanding how bacteria convert nitrate to nitrogen gas could eventually lead to effective controls of this process, thereby resulting in substantial savings to farmers. It might also be possible to turn the tables and exploit this process to safely remove nitrate from drinking-water supplies, where it can be a problem.

Copper-containing amine oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines. Amine oxidases may have a variety of functions in the cardiovascular, gastrointestinal, and nervous systems of mammals. Amine oxidases are also responsible for the cross-linking of connective tissue structural proteins (elastin and collagen). It appears that numerous compounds with antifungal; antiprotozoal, or anticancer activities may target amine oxidases; for example pentamidine,a leading drug for the treatment of Pneumocystis carinii pneumonia (PCP) in AIDS patients belongs to class of compounds that inhibit amine oxidases. The principal goals are to determine the 3-D structures of multiple amine oxidases, including at least one human enzyme, and to elucidate the mechanisms of amine oxidation and cofactor (TPQ) biogenesis. In addition, the structure and biosynthesis of a related enzyme, galactose oxidase, will be examined. Site-directed mutagenesis, spectroscopy, kinetics measurements, and crystallography are employed. The crystal structures of two amine oxidases have been solved, revealing several novel features, including a second metal-binding site. Combining structural and mechanistic data WI permit a detailed understanding of the structure and function of these important enzymes to be developed.

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.

Project Summary

This institution-wide reform project comprises three vital initiatives that enable Montana State University-Bozeman to move from a campus with promising "hot spots" of innovation in undergraduate SME&T education, to an institution with truly campus-wide involvement in these efforts. The first initiative creates a Teaching & Learning at MSU (TL - MSU) Start-Up Program to provide professional development and mentoring support to recent hires in SME&T fields. New hires will be given incentives to form a teaching & learning support team with an experienced faculty member and a graduate student to participate in a variety of professional development activities. The second initiative creates a Teaching & Learning Campus Profile for campus-wide formative assessment of present and future reforms. This crucial resource aggregates qualitative and quantitative data to provide an accurate understanding of accomplishments and needs. The third initiative creates a Faculty Teaching & Learning Portfolio (TLP) model to support meaningful evaluation of the significant professional commitment many faculty are making to improve the scholarship of teaching and learning. MSU faculty have stepped forward to meet the challenge presented by calls for fundamental rethinking of the standard teaching and learning paradigm. This project provides a much needed evaluation tool engendering the richness of faculty teaching/learning efforts and redirecting attention to student learning--the ultimate goal and test of reform. Through these three initiatives startup program that brings teams of new faculty, experienced faculty, and graduate students together to improve teaching and learning; a strong assessment component to underpin reform efforts; and a new faculty evaluation model that facilitates rewarding the practice and scholarship of teaching this proposal creates tools for MSU to use and to share with other campuses nationally.

This Integrative Graduate Education and Research Training (IGERT) award supports the establishment of a multidisciplinary graduate training program of education and research on the structure and function of complex biological systems. The program will focus on integrating knowledge and developing models of biological systems across organization levels and at multiple spatial and temporal scales. Systems to studied range from macromolecular complexes to networks of interacting nerve cells. Training will emphasize understanding complex biological systems in terms of the structures and interaction of their components, and will integrate advanced computational and mathematical approaches with a wide variety of experimental and analytical techniques. Key educational aspects are: two new, year-long, multidisciplinary courses for all students; lab rotations with a training focus, seminars in professional development and ethical practices of science, dual advisors, student involvement in collaborative, multidisciplinary research projects, a dedicated research seminar series, and intensive summer workshops led by investigators from other universities and industry. A highly productive group of faculty will participate, collectively spanning eight departments and three colleges of Montana State University-Bozeman. In conjunction with existing, successful MSU programs, special efforts are planned to increase the number of Native American students pursuing doctoral degrees in the biological sciences.

IGERT is an NSF-wide program intended to facilitate the establishment of innovative, research-based graduate programs that will train a diverse group of scientists and engineers to be well-prepared to take advantage of a broad spectrum of career options. IGERT provides doctoral institutions with an opportunity to develop new, well-focussed multidisciplinary graduate programs that transcend organizational boundaries and unite faculty from several departments or institutions to establish a highly interactive, collaborative environment for both training and research. In this second year of the program, awards are being made to twenty-one institutions for programs that collectively span all areas of science and engineering supported by NSF. This specific award is supported by funds from the Directorates for Biological Sciences, for Computer and Information Science and Engineering, and for Education and Human Resources.

The project addresses basic questions regarding the mechanism of assembly of the novel copper sites in the enzyme nitrous oxide reductase, and their roles in catalysis. Proteins encoded by the nos cluster genes nosD,F,Y,L, which are required for the biosynthesis of nitrous oxide reductase, will be purified and characterized. The investigator's hypothesis is that one or more of the Nos proteins are involved in the assembly of the novel copper cluster that is the catalytic site in nitrous oxide reductase. Over-expression methods for nitrous oxide reductase and other nos proteins have been developed. Site-directed mutagenesis, together with spectroscopic and kinetics methods, will be used to probe the structure, bonding, and reactivity of the electron-transfer (CuA) and catalytic sites. Structural studies (by NMR or x-ray crystallography) of the proteins are planned. The results should be applicable to several major themes in modern biochemistry such as metalloprotein structure and function, the assembly of metal ion clusters in proteins, and the recognition and activation of kinetically-inert small molecules in metabolic pathways.

The enzyme that is the focus of the research is the terminal enzyme in the denitrification pathway of bacteria. The denitrification pathway is a part of the global nitrogen cycle and balances the cycle by returning fixed nitrogen to the atmosphere. Global agricultural productivity and water quality are directly affected by the biological processes of nitrogen fixation, assimilation by organisms, and denitrification. Furthermore, under some circumstances denitrification may release nitrous oxide to the atmosphere, thereby contributing to ozone depletion and global warming. Consequently, it is very important to understand exactly how organisms carry out denitrification, and how to control this process.

Copper-containing amine oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines. Amine oxidases may have a variety of functions in the cardiovascular, gastrointestinal, and nervous systems of mammals. Amine oxidases are also responsible for the cross-linking of connective tissue structural proteins (elastin and collagen). It appears that numerous compounds with antifungal, antiprotozoal, or anticancer activities may target amine oxidases. For example pentamidine, a leading drug for the treatment of Pneumocystis carinii pneumonia (PCP) in AIDS patients belongs to class of compounds that inhibit amine oxidases. A major goal are to determine the 3-D structures of multiple amine oxidases, including human kidney diamine oxidase, which has been over-expressed and purified to homogeneity. Other major goals are to define the molecular bases for substrate specificity and selective inhibition among amine oxidases, and to elucidate the mechanisms of amine oxidation and cofactor (TPQ) biogenesis. In addition, the structure and biogenesis of a related enzyme, galactose oxidase, will be examined. Site-directed mutagenesis, spectroscopy, kinetics measurements, and crystallography are employed. Combining structural and mechanistic data will permit a detailed understanding of the structure and function of these important enzymes to be developed.

This research focuses on the biochemistry and structural biology of the key enzyme in the denitrification pathway. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release N2O to the atmosphere, thereby contributing to ozone depletion and global warming. Hence there exist clear and direct linkages between basic research on the biology and biochemistry of denitrification and numerous issues of substantial societal interest. Specifically, studies of the structure, mechanism, and metal cluster assembly in nitrous oxide reductase will be conducted. This research will directly address questions regarding the mechanism of assembly of the novel copper sites in nitrous oxide reductase, and their roles in catalysis. Periplasmic proteins coded by the nos cluster genes nosD, L, and X (which are involved in the biosynthesis) and nitrous oxide reductase (coded by nosZ), will be purified and characterized. Investigation of cluster assembly in vitro will be conducted with purified nitrous oxide reductase and other nos proteins. Functions of the nos proteins in vivo will be probed by the generation and characterization of knockouts. Site-directed mutagenesis, together with structural, spectroscopic and kinetic methods will be used to probe the structure, bonding, and reactivity of the electron-transfer catalytic sites. Mechanistic, spectroscopic, and structural studies of the reductively-activated form of nitrous oxide reductase will be emphasized owing to the recent demonstration that this form is catalytically competent.

Broader Impact: Undergraduate and graduate students will be directly involved in the research project, including students from baccalaureate colleges and groups underrepresented in science. Further, the research is conducted as part of MSU-Bozeman's IGERT program in Complex Biological Systems and therefore may contribute to institutional improvement in graduate education. Given the importance of the global nitrogen cycle and denitrification, there will be numerous opportunities (both formal and informal) to present the linkages between basic research and issues of broad interest and importance to society.

DESCRIPTION (provided by applicant): We propose a phased program for the renovation of the Cooley Microbiological Laboratories at Montana State University (MSU). The laboratories consist of four floors and a basement and are highly inefficient, with no centralized cooling, air supply, vacuum, or distilled/RO water. They are also cited by the Bozeman Fire Department every year for infrastructure and overuse violations. In this proposal, we are requesting funds from the NIH for the first phase of the renovation program. This phase addresses the complete renovation of the top two floors (Floors 3 & 4) for our biomedical research group. In addition, a new elevator is proposed to meet current federal and local code requirements, and partial renovation of the basement is proposed to allow installation of utilities to the building, including cooling requirements for a shared genomics facility and bioinformatics research and training center. In order to meet current and future needs for biomedical research, our specific aims include: . Removal of all existing walls and corridors to be replaced with open plan modular laboratory designs using the Max/Lab(tm) Adaptable Furniture System or equivalent. . Increase the current laboratory areas by approximately 13% and allow for far greater efficiency in interactions between faculty, research staff and students. . Include a 300-square-foot BSL-3 laboratory to be used by research programs from all floors of the Cooley Laboratories, from other MSU departments and as a resource for the State of Montana at times of disease outbreak emergencies. . Include faculty and student offices, equipment rooms, a tissue culture room, microscope room, walk-in cold rooms, electrical closets, chemical dispensing rooms, break rooms and new ADA compliant restrooms.

DESCRIPTION (provided by applicant): Copper-containing amine oxidases and lysyl oxidases are widely distributed in nature and are involved in the metabolism of biogenic primary amines, in the maturation of connective tissue, and numerous other physiological processes. The human vascular adhesion protein (hVAP-1) is an amine oxidase, and its oxidase activity is directly involved in cellular adhesion. Multiple "lysyl oxidase like" (LOXL) proteins have been recognized in mammals, and these proteins may substitute for lysyl oxidase, but may also have roles in multiple cellular processes, including differentiation, proliferation, and motility. It appears that recently recognized biogenic amines and several pharmaceutical compounds may be metabolized by or inhibit amine oxidases. A multidisciplinary approach emphasizing spectroscopy, kinetics, site directed mutagenesis, and crystallography is proposed to elucidate the chemical and biological principles that define key structure- function relationships in amine oxidases and lysyl oxidases. A major goal is to determine the structures of key amine oxidases, including a lysyl oxidase, and of amine oxidase and lysyl oxidase complexes with inhibitors and substrate analogues. Other major goals are to define the active-site structures, catalytic mechanisms, and the molecular bases for substrate specificity and selective inhibition in amine oxidases, including hVAP-1 and LOXLs. Experiments designed to resolve outstanding mechanistic questions in the biogenesis of TPQ and LTQ are also proposed. In addition, the structure and biogenesis of a related enzyme, galactose oxidase, will be examined. Combining structural and mechanistic data will permit a detailed understanding of the structure and function of these important enzymes to be developed.

This research focuses on the biochemistry and structural biology of the key enzyme in the denitrification pathway. Denitrification is an intrinsic part of the global nitrogen cycle and is the pathway that balances the cycle, returning fixed nitrogen to the atmosphere. The environmental biology of nitrogen fixation, assimilation, and denitrification substantially impacts agricultural productivity and water quality. Denitrification may release nitrous oxide (N2O) to the atmosphere, thereby contributing to ozone depletion and global warming. Hence, there exist clear and direct linkages between basic research on the biology and biochemistry of the denitrification pathway and numerous issues of substantial societal interest. Specifically, this research is concerned with the structure, mechanism, and assembly of the key enzyme in denitrification, nitrous oxide reductase. In the vast majority of denitrifying organisms, nitrous oxide reductase is the terminal enzyme in the denitrification pathway. The research will investigate the structures and reactivities of the novel copper sites in nitrous oxide reductase, and their roles in catalysis. Detailed experimental studies of proton-transfer and electron-transfer steps in the reduction of N2O will be carried out. The structure of the fully reduced form of the enzyme, which reacts directly with substrate, will also be pursued. An additional protein, named NosX, also appears to play a key role in the reduction of N2O. The hypothesis is that NosX activates the reductase in cells and also regenerates enzyme that has become inactivated. This hypothesis will be thoroughly tested by several approaches. Successful over-expression methods for nitrous oxide reductase and NosX have been developed and will be exploited to prepare proteins for study. Site-directed mutagenesis, together with spectroscopic and kinetics methods will be used to probe the structure, bonding, and reactivity of the catalytic sites in nitrous oxide reductase and NosX. Structural studies of the proteins will be carried out by nuclear magnetic resonance (NMR) or X-ray crystallography. Both undergraduate and graduate students will be directly involved in the project. The context and significance of the project is of broad public interest, and will be presented and featured in multiple venues by the Principal Investigator.

Broader Impacts. The Department of Chemistry and Biochemistry at Michigan State University is a NSF Research Experiences for Undergraduates (REU) site, and the PI is a member of the participating faculty. An important aim of the REU program is to provide research opportunities for Native American students and students from other underrepresented groups. It is anticipated that one or two undergraduates (annually) will be involved in this research as part of their participation in the REU program. In addition, this project may support the participation of students from Rocky Mountain College in Billings, Montana, an undergraduate college with extremely limited opportunities for undergraduate research. The research on denitrification, with its clear connections to agriculture and to environmental issues, has served as an effective focal point for informal presentations and discussions on the relationships and linkages between basic research and benefits to society. The clear connections between the denitrification pathway and environmental, agricultural, and energy issues are regularly emphasized in talks to prospective students, undergraduate and graduate students, community groups, and interest groups. The Principal Investigator, who also serves as the Provost and Vice President for Academic Affairs at MSU-Bozeman (Montana's leading research institution and a Carnegie "very high research activity" university), has many opportunities annually to speak to the importance of research and discovery to undergraduate education and to economic development in the state. He meets frequently with members of state government, legislators, and numerous constituencies.