Frank Jordan - US grants

The long term goal of this proposal is the elucidation of the structure and mechanism of action of thiamin diphosphate- dependent pyruvate decarboxylating enzymes; pyruvate decarboxylase and pyruvate dehydrogenase complex. The latter enzyme is at a key junction in metabolism; a thorough understanding of its structure and mechanism is of fundamental biochemical importance. The enzymes will be purified in part by immunoaffinity chromatographic methods. The specific objectives are to: 1. design and synthesize mechanism-based inhibitors identify the sequence of the coenzyme and substrate binding sites and determine the location (including surrounding sequence) of the key Cys SH's of the enzymes. 2. determine the structure of the key enzyme-bound enamine using electronic spectroscopy. Ascertain its reactivity vis-a-vis protonation, disulfides, and flavin, in reactions mimicking all the important classes of thiamin diphosphate-dependent alpha-keto acid decarboxylating enzymes.

This proposal;l is concerned with delineation of the remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C.4.1.1.1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewers'yeast enzyme was purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the AI and collaborators at the VA Hospital in Pittsburgh. A 2.4 electron density map of this enzyme is more than 95% complete, including the coenzyme binding site. With this map already in hand, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in some of the three putative covalent substrate-coenzyme complexes; b. the activation towards catalysis (in the absence and presence of substrate activation) of the two aromatic rings (thiazolium and 4-aminopyramidine), and test of the hypothesis that both rings (not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis, for example determining if surrounding hydrophobic amino acids provide a "solvent effect" thereby lowering the transition state energies of a number of key steps, as suggested by model studies ; and d. structural bases and structural as well as mechanistic consequences of substrate activation (using among other tools pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form). Most prominent among the tools to address these questions will be (all of these are "in hand" as of writing): a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh); b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; 4. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to compare the three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research will be of profound interest and significance to many other research groups working on thiamin around the globe.

Thiamin diphosphate (TDP, the vitamin B1 coenzyme) is one of the most important coenzymes in carbohydrate metabolism catalyzing e.g. the oxidative decarboxylation of pyruvate to acetylCoA in the reaction catalyzed by the pyruvate dehydrogenase multienzyme complex, enabling the product of glycolysis to enter the citric acid cycle. High resolution structural of such TDP-dependent pyruvate decarboxylating enzymes are being conducted by a variety of techniques. The principal target has been brewers'yeast pyruvate decarboxylase (PDC, E.C. 4 1.1.1) recently purified as fully active alpha4 and beta4 homotetramers in addition to the alpha2beta2 heteroteramer reporter earlier. In a most important and exciting recent development the alpha 4 structure was crystallized in the laboratory already providing novel information, and pointing to the likely success of resolution to at least 2.4 A. PDC is the most widely studied TDP-dependent eznyme and is therefore the best one on which to undertake the detailed structural studies proposed below. In addition to the x-ray developments major progress has been made in the spectroscopic characterization of enzyme-bound intermediates. It is proposed to extend these studies along the following lines of inquiry: 1. structural work on PDC by x-ray crystallographic methods (this part of the research is a collaborative effort with SAX, Fuery and coworkers at the VA Hospital Biocrystallography Lab/Univ. of Pittsburgh)n to determine: a. the structure of the "TDP fold", identify the putative beta-turn-alpha-turn-beta structure at the TDP binding site; b. the basis of interaction between the TDP and Mg2+ with PDC; c./ the conformation of bound TDP, and of putative covalent intermediates; d. the nature of protein-protein interactions in PDC, i.e. among subunits in the absence and presence of the allosteric regulator pyruvamide; 2. mechanistic work of PDC, especially to elucidate the nonoxidative and oxidative chemistry of the enzyme-bound enamine; 3. immunochemical structural studies on PDC to determine immunochemical differences, if any, among isoforms; and on pyruvate dehydrogenase to probe its structure with the monoclones already at hand; 4. structural work on PDC and its TDP fold by NMR methods to determine the microenvironmental factors in catalysis. A collaboration has been initiated with S. Hohmann, who has cloned PDC in a variety of forms, and has and will supply the yeast containing the site-directed mutants, as well as the clones to address both structural and mechanistic questions.

This proposal is concerned with delineation of remaining structural and mechanistic questions on pyruvate decarboxylase (PDC, E.C. 4. 1. 1. 1), perhaps the simplest nonoxidative decarboxylase requiring thiamin diphosphate (ThDP, the vitamin B1 coenzyme, of fundamental significance in human metabolism). The brewer' yeast enzyme was recently purified as fully active alpha4 and beta4 homotetramers and the alpha4 structure was crystallized by the PI and collaborators at the VA Hospital in Pittsburgh pointing to the likely success of resolution to at least 2.4 angstroms. With the evidence already accumulated from the 3 angstroms map of the enzyme, for the first time on this representative of a large group of alpha-keto acid decarboxylases, rational experiments can be designed to probe: a. the conformation of the enzyme-bound coenzyme in the absence of substrate, as well as in the three covalent substrate-coenzyme complexes invoked, based on chemical analogy; b. the chemical behavior in the absence and presence of substrate of the two aromatic rings (thiazolium and 4-aminopyrimidine), and test of the hypothesis that both rings ( not only the thiazolium) are participants in catalysis; c. the environment of the coenzyme and the function of amino acids surrounding it in catalysis; and d. the effect of pyruvamide, a nondecarboxylatable substrate surrogate that is capable of shifting the enzyme from a low to a high activity form, on structure and mechanism. Most prominent among the tools to address these questions will be : a. x-ray crystallographic methods (this part of the research is a collaborative effort with Furey, Sax and coworkers at the VA Hospital Biocrystallography Lab/ Univ. of Pittsburgh) ; b. site-directed mutagenesis of amino acids found near the catalytic and regulatory sites based on the x-ray crystallographic and mechanistic information; c. further elucidation of the chemistry and enzyme-bound environment of the enamine, one of the three ThDP-substrate covalent complexes on PDC; d. multinuclear magnetic resonance on ThDP bound to PDC, with ThDP labelled at the C2, Nl' and N4' atoms to provide information concerning the state of ionization, and the charge densities of the coenzyme during the catalytic sequence in the absence and in the presence of regulators;. and 5. modeling based on the x-ray coordinates to help refine mechanistic models, to help design even more insightful experiments, and to help construct three dimensional models of two enzymes with very high sequence homology to PDC's (pyruvate oxidase and acetolactate synthetase) that have not been crystallized yet, and that have identical mechanisms through pyruvate decarboxylation, but diverge significantly thereafter. Undoubtedly, the fundamental questions to be resolved by the proposed research, will be of profound interest and significance to many other research groups working on thiamin around the globe.

9413198 Jordan This award to Frank Jordan and a group of 11 other faculty will support a multi-disciplinary training program in biophysical aspects of cellular processes at Rutgers University-Newark, an urban campus with significant enrollement of minority students. The faculty group have excellent research programs in three related areas: structure and dynamics of biological macromolecules, analysis of cellular transport mechanisms, and biophysics of excitable cells. The award will support training of students at the undergraduate and graduate levels; funds awarded will provide student stipends and tuition, purchase research instruments and supplies to be used by trainees, seminars by outside experts and student travel. Current training activities will be enriched by three new laboratory courses and additional required rotations in faculty laboratories. ***

This award from the Chemistry Research Instrumentation and Facilities (CRIF) Program and the Major Research Instrumentation (MRI) Program will assist the Department of Chemistry at Rutgers University to acquire an Raman spectrometer and an Ar+ pumped mode-locked Ti:sapphire laser. This equipment will enhance research in a number of areas including the following: (1) monomolecular films at the air/water interface, (2) the ultrafast kinetic investigation of excited states and reactive species, (3) synthetic proteins as models, and (4) enzyme mechanisms. A laser can provide important information about chemical reactivity. Its use may enable breakthroughs in our understanding of the properties of reactivity and non-reactivity of molecules..

This award provides funds to support the purchase of an instrument with combined liquid chromatography and advanced time of flight and tandem mass spectrometry capabilities. Recent advances in mass spectrometry have provided unprecedented opportunities for the analysis of proteins, such as internal sequencing, identification of nature and location of post-translational modifications and noncovalent interactions. The system to be purchased will allow the research communities at the University of Medicine and Dentistry of New Jersey and Rutgers University - Newark to take advantage of this state-of-the-art technology in protein research. Currently, there is no instrument available that allows researchers at either institute to sequence and elucidate protein structures using tandem mass spectrometry. Provision of this technology will significantly enhance the quality and productivity of the research on both campuses, as well as at others in the Newark area. The instrument will be placed in a Mass Spectrometry Core Facility available to researchers at both institutions. The projects outlined in this application by the expected major users of the instrument entail basic research into the structure and function of proteins from a wide-range of organisms - human, yeast, bacteria and plants. The types of questions to be addressed will require protein identification and sequencing, nuclear protein complex identification, localization of protein-ligand interactions, enzyme tertiary structure determination, quality control of designer metallo-protein synthesis, internal sequencing of N-terminally blocked proteins, and identification of post-translational modification of proteins. This equipment will be supervised and maintained by an experienced mass spectrometrist who is in charge of the core facility. Three committees drawn from both institutions oversee the fiscal and scientific management and daily operation of this facility.

DESCRIPTION (adapted from applicant's abstract): The overall objective of this research is to enhance our understanding of structure-function relationships, including the mechanism and regulation of the El component of the pyruvate dehydrogenase multienzyme complex isolated from E. coli. This enzyme requires thiamin diphosphate (the vitamin B coenzyme) and decarboxylates pyruvate, the product of glycolysis, to initiate a series of reactions, a major product being acetyl-Coenzyme A, the starting material for the citric acid cycle. Among the specific goals for the requested period are: (1) Production of wild-type and variant El enzymes (the variants being made by site directed mutagenesis of the plasmid specifically bearing only the gene coding for El) for both mechanistic and structural studies, including, determination of the X-ray structure in a collaboration wit William Furey, University of Pittsburgh (with whom the principal investigator has collaborated in the solution of the simpler yeast pyruvate decarboxylase structure); (2) Experiments designed to a. identify the amino acids responsible for catalysis, and assign the function in catalysis; b. study the fate of key thiamin-bound intermediates on the enzyme; c. study the rate-limiting steps in catalysis and the structure of transition states in wild-type and active center variant enzymes; d. identify the site of, and solve the mechanism of inactivation of El by substrate fluoropyruvate, 2-oxo-3-butynoic acid (a potent substrate analog inhibitor developed in the principal investigator's laboratory), a putative transition-state analog, and several highly inhibitory monoclonal antibodies (also developed in the principal investigator's laboratory); (3) Experiments to identify the site(s) at which a variety of metabolic regulators interact with the El, including the cofactors and the products of the reaction, as well as GTP- and the pathway by which the information i transmitted to the active center; and (4) Explore the principal investigator's hypothesis concerning the key reductive acetyl transfer between the El and E2 enzymes. Since this enzyme is at a key metabolic junction, the principal investigator believes that in the coming years he and his collaborators have an opportunity to contribute to a better molecular-level understanding of this member of a very large class of related multienzyme complexes.

A grant has been awarded to Rutgers University under the direction of Dr. Jean Baum to purchase an 800 MHz NMR spectrometer as part of an initiative to develop a New Jersey State-Wide 800 MHz NMR Facility. The 800 MHz NMR will initially serve a minimum of four New Jersey institutions including Rutgers-New Brunswick (NB), Rutgers-Newark, Princeton University and Montclair State University (MSU). Ten faculty members, three from Rutgers-NB/RWJ, one from Rutgers-Newark, four from Princeton and two from MSU are participating in the New Jersey Wide 800 MHz NMR Facility. Access to an 800 MHz NMR will allow the State-Wide group to implement a range of state of the art biological NMR experiments that are currently not feasible on the 500 and 600 MHz NMRs and will enhance their productivity in terms of time intensive NMR projects.

Establishment of the NJ State-Wide 800 MHz NMR Facility will enhance the quality and accessibility of NMR research and education to the NJ community. State of the art NMR research which cannot presently be performed on the lower field instruments that exist at the respective individual institutions will now be accessible to the New Jersey NMR community. In addition, the NJ-State wide 800 MHz NMR facility provides an opportunity for undergraduates, graduates and postdocs to meet researchers from other New Jersey schools, to be trained on state of the art NMR instrumentation and to be exposed to many different NMR research projects. Having the 800 MHz NMR serve a number of different institutions provides an intellectual environment and collaborative framework for NMR users. 'Hands on' NMR experiments will be performed by approximately 60 graduate students, postdocs as well as undergraduates involved in NMR research. The research activities of the members of the facility are in molecular biophysics with emphasis in the following areas: Protein structure determination; Conformational studies and dynamics of folded and unfolded proteins to understand the basis of protein folding and misfolding; Studies of nucleic acid structure and interactions; NMR investigations of protein-protein and protein-nucleic acid interactions; Design and conformational analysis of small peptides; NMR methods development for next generation application.

The New Jersey Statewide 800 MHz NMR facility that we will establish will provide infrastructure and enhance the quality and accessibility for many research and educational programs at both private and public Universities in New Jersey. In bringing together this group of Universities, we are establishing an environment of collaboration and are coordinating expertise in NMR from many different institutions. The NJ wide 800 MHz NMR facility provides an opportunity for undergraduates, graduates and postdocs to meet researchers from other New Jersey schools and to be exposed to many different NMR research projects. Having the 800 MHz NMR serve a number of different institutions provides an intellectual environment and collaborative framework for NMR users.

DESCRIPTION (provided by applicant): This is a request for renewal of a research program on thiamin diphosphate-dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry to the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project are: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. In innovation requiring de novo total synthesis of specifically labeled thiamins, the PI proposes to use solid state NMR, as well as advanced solution NMR methods to accomplish the goals. (2) Exploration of the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. With methods published by the PI in 2008, key loops on the E. coli complex's E1 and E2 components have been identified for these studies, including the lipoyl domain of the E2 component, which 'visits' the active centers of all three components. (3) Examination of the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognizes the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, we wish to determine the regions of E2 component interacting with the E1 component, never accomplished before The methods used by the PI could answer questions that no other current methodology can, such as the definition of proton positions on the 4'-aminopyrimidine ring of the coenzyme at distinct intermediates along the reaction coordinate, a key issue in understanding proton transfers. A group of outstanding collaborators have been recruited for experimental expertise not available in the PI's laboratory, among them M. Patel, a CoPI selected for expertise on the human enzyme, and W. Furey, a long-term collaborator in a string of structure determinations. Results from two complexes will enable the PI to draw general conclusions regarding the entire superfamily of such enzymes, as already demonstrated in several of his recent publications.

With this award from the Major Research Instrumentation (MRI) and support from the Chemistry Research Instrumentation and Facilities (CRIF) Programs Professor John Sheridan from Rutgers Universvity Newark and colleagues Frank Jordan, Elena Galoppini, Frieder Jaekle and Darren Hansen will aquire a 500 MHz NMR spectrometer. The proposal is aimed at enhancing research training and education at all levels, especially in areas such as (a) development of functional conjugated macrocycles and polymers for optoelectronic applications and synthesis of chiral Lewis pairs for small molecule activation; (b) investigation of stimuli-responsive metal-containing polymers; (c) synthesis of functional molecules for dye-sensitized solar cells (DSSCs), host-guest complexes, and biosensors; (d) development of sustainable polymers from renewable resources; (e) investigation of thiamin diphosphate (ThDP)-dependent enzymes as well as synthesis of labeled coenzymes, substrate analogues and inhibitors; and (f) mechanistic studies on quinolone quorum sensing.


Nuclear Magnetic Resonance (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. Access to state-of-the-art NMR spectrometers is essential to chemists who are carrying out frontier research. The results from these NMR studies will have an impact in synthetic organic/inorganic chemistry, materials chemistry and biochemistry. Many research projects associated with this application will have a direct impact on society, as they aim to develop polymers fo optoelectronics, metal containing polymers, materials for solar cells, and investigations of enzyme inhibitors and bio-sensing. This instrument will be an integral part of teaching as well as research at Rutgers University Newark.

Because life at the molecular level is the chemistry of carbon containing compounds, reactions that make carbon-carbon bonds are particularly valuable. A challenge for making compounds from carbon is the formation of closely related versions or isomers as products from a single reaction. The most closely related isomers are called stereoisomers, otherwise identical compounds, but mirror images of each other. The proposal envisions harnessing the power of biological transformations for carbon-carbon bond formation that make single stereoisomers for the production of practical compounds like pharmaceuticals. The reactions would not only be exquisitely specific, but also environmentally friendly, or "green" by using only natural materials and gentle conditions. The research plan involves the re-engineering of proteins using recombinant DNA technology so that they specifically make high-value products. The projects that make up the plan will enable interdisciplinary training of graduate students, postdoctoral fellows and undergraduates in the fields of enzymology, molecular biology, protein engineering, and chemical synthesis.


With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Frank Jordan and Edgardo T. Farinas, of Rutgers University-Newark Campus and the New Jersey Institute of Technology, respectively, to undertake enzymatic synthesis of chiral alpha-hydroxy ketones for pharmaceuticals, and fine chemicals for synthesis of larger molecules. For the first time the 2-oxoglutarate dehydrogenase multienzyme complex (OGHDc), both human and E. coli, and 2-hydroxy-3-oxoadipate synthase from Mycobacterium tuberculosis (HOAS), will be engineered to create these compounds. The principal investigators have already demonstrated that the first OGDHc component (E1o) is an efficient biocatalyst using the physiological substrate. To enhance the power of this carboligation reaction, they will: (1) Engineer residues from the substrate binding site selected from X-ray structures of the E1os and HOAS to accept a variety of substrates for efficient enzymatic synthesis of alpha-hydroxy ketones, with high enantiomeric excess (ee) and yield; (2) Explore alterations in the chemical nature of donor 2-oxo acid and of acceptor aldehyde. (3) Advance the field of biocatalysis by engineering not only E1os but the second E2o core component of OGDHc to accept unnatural substrates leading to acyl-coenzyme A analogues participating in many metabolic pathways; (4) Explore the mechanism of intra-E2o component acyl transfer to coenzyme A.

? DESCRIPTION (provided by applicant): The PIs goals with this R15 proposal are studies on the thiamin diphosphate (ThDP)-dependent human 2-oxoglutarate dehydrogenase complex (OGDHc, also known as ?-ketoglutarate dehydrogenase; it comprises three component enzymes denoted with E1o-h, E2o-h and E3-h), the rate-limiting enzyme in the citric acid cycle (also known as the tricarboxylic or Krebs cycle), an enzyme critical for glucose metabolism in normal brain. OGDHc activity is diminished in the brain of Alzheimer's disease patients, which is characterized by reduced glucose metabolism and increased level of oxidative stress markers. A diminished OGDHc activity had also been correlated with other neurodegenerative diseases, including Parkinson's disease, Huntington disease, Wernike-Korsakoff disease and progressive subnuclear palsy. It is known that OGDHc is a sensitive target for a variety of oxidants generated during oxidative stress, including inhibition of OGDHc by peroxynitrite and nitric oxide, and by the reactive oxygen species (ROS), superoxide and H2O2, among other agents. OGDHc is not only a sensitive target of ROS, it can also produce superoxide and H2O2 in brain mitochondria. While earlier research attributed ROS generation by OGDHc to the E3-h component, the PI and collaborators have now shown definitively that (a) The ThDP-dependent E1o-h component in fact also produces superoxide anion and hydrogen peroxide, (b) Concomitantly, there is produced a ThDP-bound radical characterized by electron paramagnetic resonance spectroscopy (EPR). These products result from reaction of a ThDP-bound intermediate with dioxygen O2. Our contributions, and this proposal were made possible by the PI's group's recent success in producing full length, active recombinant versions of the human E1o-h and E2o-h components of this important complex in pure form and in significant amounts for the proposed research, while other groups address this issue mostly on intact brain mitochondrion, rather than with individual protein components of the human OGDHc. The OGDHc-h is located in the matrix of the mitochondrion where it is associated with the inner mitochondrial membrane, catalyzing the conversion of 2-oxoglutarate to succinyl-CoA according to: 2-oxoglutarate+NAD++CoA ? succinyl-CoA+NADH+H++CO2 (1) The overarching goal of our studies is an atomic-level explanation of: Which component (E1o-h and E3-h) is the major source of ROS production, consistent with the medical finding of reduced OGDHc activity in the brain with aging. Our long term goal is to provide an atomic explanation for the ROS findings in brain by identifying specific residues responsible for ROS formation, and those modified by ROS on all three components of the human OGDHc. Our immediate goals within the boundaries of the R15 program, are to initiate a logical sequence of projects, which will lead to achieving of our long-term goals, include: (a) Detection and kinetic characterization of thiamin-related intermediates on E1o-h and lipoamide-bound intermediates on E2o-h using a battery of kinetic and spectroscopic state-of-the-art methods developed in our group, as well as new methods EPR and ROS detection. We will determine the effect of OGDHc-h assembly, and of the contribution of the three individual components of OGDHc-h to ROS generation. No such studies are yet done on the human OGDHc. The mechanistic significance of these studies is to provide an understanding of how and where the ThDP-enamine radical formed by reaction of dioxygen with the enamine, receives special stabilization on the E1o-h; and which residues of E1o-h are the major contributors to ROS generation by OGDHc-h. (b) Development of an advanced laboratory course titled Introduction to Modern Biochemical Research to train both undergraduate and graduate students in state-of-the-art methodologies, with experiments strictly paralleling those proposed for the research program, and with the enzymes studied in our program; the tools to be taught will include, kinetics, spectroscopy (circular dichroism, MS, NMR, EPR), and basic tools of molecular biology and biochemistry. No such course exists at any Rutgers campus in Newark, hence it will serve the needs of both arts and sciences and the medical school in Newark. This course will also enable the Chemistry Department to at last offer a Biochemistry track; such a lab course is also highly recommended by the Am. Chem. Society. Significance and Innovation. An understanding of the mechanism by which OGDHc produces ROS, and how OGDHc activity could be altered by ROS is an important goal of the PI since it could provide insight to neurodegenerative diseases. This is the gap in our knowledge that our proposed research will help to fill. Methods for all experiments in the Specific Aim are in place i the PI's laboratories and are in published material, hence the proposed experiments carry little risk. Gary Gerfen at Albert Einstein College of Medicine has agreed to collaborate on the EPR studies. The close relationship between the course being proposed and the research being proposed is surely innovative and is in the spirit of the R-15 program.