Roderick A. Capaldi - US grants

The ultimate goal of the research proposed here is to elucidate the mechanisms of ATP hydrolysis and ATP synthesis by the ATP synthase, an enzyme common to all energy transducing membranes. At present, the information obtained on the structure of the ATP synthases in mitochondria chloroplasts and bacterial plasma is fragmentary. The enzyme from all sources consists of an F1 portion, outside the bilayer, made up of five or in some cases six different subunits, including the inhibitor protein. The F1 is bound to a membrane intercalated part, the FO portion, which is made up of three different subunits in the bacterial and chloroplast enzyme but more in the mitochondrial enzyme. The 5 subunits of F1 in Escherichia coli and beef heart mitochondria are present in the stoichiometry of Alpha, 3; Beta, 3; Gamma, l; Delta, 1; Epsilon; 1. The F1 contains three active sites that can work independently but only slowly. In the presence of an excess of substrate to enzyme, the F1 ATPase functions cooperatively and hydrolyzes ATP by a factor of 2 thousand to a million times faster than in single-site catalysis. Our immediate goals are to examine whether the three catalytic sites are structurally and functionally the same or not as well as to locate the active sites in the protein and identify residues critical to the observed cooperativity between these sites. Other structural studies are planned, including experiments to identify subunits of FO that bind the F1; experiments on the orientation of F1 with respect to the membrane and experiments to characterize the environment of a critical carboxyl in the FO, i.e. the DCCD-binding glutamic acid (Asp 61 in subunit c of Escherichia coli). Longer term studies will include attempts to crystallize both F1 and FO and experiments on the conformation of F1 and FO, as well as estimation of functionally important conformational changes in the ATP synthase.

The mitochondrial respiratory chain is responsible for the conversion of the chemical energy in the breakdown products of foodstuffs into chemiosmotic energy for use in ATP synthesis. There are four multicomponent complexes making up the respiratory chain, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV). Cytochrome c oxidase is composed of a 9 different subunits in fungi such as the yeast Saccharomyces cerevisiae and 13 different subunits in mammals, 3 coded for on mtDNA and made in the mitochondrion, the remainder encoded in the nucleus. A low resolution structure of the mammalian enzyme has been obtained (25 Angstroms). Ongoing work is aimed at localizing subunits within this structure and determining the orientation of each subunit. Recent evidence indicates that several of the subunits of the mammalian cytochrome c oxidase are present as isoforms. Studies are proposed to characterize the isoforms of the enzyme complex in beef and in humans and to determine the tissue distribution of these forms. An analysis of the tissue specificity and developmental regulation of the isoforms of cytochrome c oxidase should greatly aid in understanding the molecular basis of a variety of human diseases due to dysfunction of this enzyme. Other projects proposed involve crystallization of cytochrome c oxidase using enzyme from a variety of sources and establishment of the molecular basis of cytochrome c oxidase deficiency in a case of Kern-Sayres syndrome, preliminary study on which suggest the defect involves splicing of the subunit IV transcript.

Funds are requested as support for travel and 5 days housing and subsistence of 27 key speakers or discussion leaders plus room and board only for 25 other participants at the 1985 Gordon Conference on Energy Coupling Mechanisms. This meeting is the only opportunity for American Bioenergeticists to meet and exchange ideas. The proposed program will have 22 invited lectures. All participants (150) will be encouraged to present posters and the daily display of posters will be discussed in open forum as part of the evening sessions. In this way emphasis will be placed in promoting the maximal scientific interchange between established investigators and the many young scientists that will be invited. The program will consist of eight major sessions as follows: 1) Mitochondrial Electron Transfer Chain: Structure and Function; 2) Electron Transfer Chains in Bacteria; 3) Structure and Function of Photosynthetic Reaction Centers; 4) The ATP Synthase: Mechanistic Considerations; 5) ATP Synthase: Structural Considerations; 6) Structure and Function of Bacterial Transport Proteins; 7) Proton Pumping ATPases in Different Organelles; 8) Mitochondrial Myopathies. The Gordon Conference on Energy Coupling Mechanisms consists of one work week of total immersion, discussion and information exchange among 150 international investigators. The funds requested will help insure the participation of the key speakers, many from Europe, and will provide support for junior scientists to enable them to attend the conference at a time when interchange of scientific ideas is critical to their career development.

This Biological Facilities Center proposal requests funds for the purchase of ancillary protein sequencing equipment, peptide synthesis and purification equipment, and equipment for x-ray structure determination of native and engineered proteins. The equipment will be shared by the staff of the Institute of Molecular Biology, one of the leading centers for the study of macromolecular structure and function, and its subsidiary program in Cell Biology. The equipment will be used to identify and/or study proteins involved in the mechanism and control of DNA replication and transcription, the effect of changes in primary structure on stability and tertiary protein structure, the interaction of subunits within protein complexes and between membrane proteins and lipids, the export of proteins by eukaryotic cells, and the mechanism of signal transduction in bacterial chemotaxis. This instrumentation will facilitate the collaborative research that has resulted in important scientific contributions in areas such as the stability of proteins, protein-DNA interactions, DNA replication and DNA transcription.

The universal fuel of cellular processes is adenosine triphosphate or ATP. In some bacteria, algae and plants, the energy stored in ATP comes from photosynthesis. In other bacteria and in all animals, the energy for ATP synthesis from ADP + Pi comes from the breakdown of foodstuffs, with the bulk of the energy transformation ocurring in a process called oxidative phosphorylation. The enzyme responsible for synthesis of ATP in photosynthesis and in oxidative phosphorylation, the ATP synthase, is a large multisubunit complex associated with the plasma membrane in bacteria, the inner membrane in mitochondria and the thylakoid membrane in chloroplasts. The ATP synthase from all of these sources is structurally similar, with a water soluble part extrinsic to the bilayer (F1 part) and transmembrane sector (FO part). Besides making ATP, synthase hydrolyzes ATP in a way that conserves the energy in the high energy phosphoryl bond for coupled transport processes. How this ATP synthase functions is unclear. To get at this question we are proposing a structure determination of the large protein complex. Our plan is to use electron microscopy of two- dimensional crystals of F1 and single particle analysis of F1 and F1F0 in combination to obtain a three-dimensional structure of the entire complex to 20-25A resolution. This low resolution structure will be used as a framework in which to locate individual subunits and functionally important sites including nucleotide binding sites.

Partial funding for the purchase of a shared 500 MHz NMR spectrometer is requested. The proposed instrument will greatly aid in the nine research projects outlined in this proposal. These include the structure and dynamics of bacteriophage T4 lysozyme; the structure of the "zinc finger" of bacteriophage T4 gene 32 protein; the nature of the interactions of tetraalkylammonium compounds with DNA; the structure of a determinant in the sorting of yeast carboxypeptidase to the vacuole; the structure and thermodynamic properties of an aspartic acid residue involved in proton pumping by the F1/F0 ATPase; an investigation of the direct transfer of substrates between enzymes; the synthesis of biologically important compounds; the mechanism of action of coenzyme B12; and the structure and mechanism of inhibition of serine proteases by selenoxide containing compounds.

This award will support a seminar on "Mitochondrial Biogenesis - Mechanism and Pathobiology," organized by Prof. Roderick A. Capaldi of the University of Oregon and Prof. Takayuki Ozawa of the University of Nagoya, Japan. Participants will meet in Honolulu, Hawaii, December 4-6, 1989 to exchange recent research results and promote future joint research projects. The seminar will deal with the biogenesis of the mitochondria, mitochondrial diseases, and in particular the role of mitochondrial DNA in human diseases. Research in this field has progressed rapidly, but is usually conducted and reported along narrow disciplinary lines. This seminar, on the other hand, will bring together biochemists, geneticists, molecular biologists, and clinicians in an attempt to find common threads, related to mitochondrial genetic variations, in the propagation of a variety of human diseases. This interdisciplinary and international discussion of some of the latest research results in this area could have important implications in medicine.

The ultimate goal of the research proposed here is to elucidate the mechanism of ATP hydrolysis and ATP synthesis by the ATP synthase, an enzyme common to all energy transducing membranes. Alterations in the ability or efficiency of these coupled reactions may be an important factor in aging and can explain some of the cases of mitochondrial myopathy, encephalopathy and cardiopathy that are being reported with increasing frequency in the medical literature. Work in this laboratory began with the bovine heart ATP synthase but has switched to the enzyme from Escherichia coli. The bacterial enzyme has many of the structural features and functional properties of the heart enzyme but can be manipulated genetically and can also be dissociated into component subunits and then reassociated into a functional complex. The major approach being used is electron microscopy of specimens embedded in a thin layer of ice (cryoelectron microscopy). A three-dimensional structure of the ECF1 has been obtained at low resolution (25A). A collection of monoclonal antibodies (mAbs) have been purified against the various subunits of the complex and these are being used to decorate ECF1 and ECF1F0. The antibody-antigen complex can then be examined by electron microscopy and the locus of individual subunits in the structure identified. The structural changes that accompany functioning of the enzyme complex are also being studied. ECF1 shows cooperative ATPase activity, i.e. binding of substrate ATP, in one catalytic site increases the off-rate of product ATP in another site. In ECF1F0, this cooperativity is important in both ATP hydrolysis and ATP synthesis, and these reactions are coupled to proton pumping through the F0 part. Protease digestion, mAb binding, chemical crosslinking and cryoelectron microscopy are all being used to examine the nature of the conformational changes that occur during cooperative ATPase activity and in coupling of this reaction to proton pumping.

F1F0-type are an important group of membrane-bound multisubunit enzymes involved in using ATP to generate a proton gradient, which can be used for substrate or ion transport. The primary role of these enzymes in mitochondria or chloroplasts in eukaroyte cells is the synthesis of the cellular fuel, ATP. Emerging evidence is that the F1F0 ATPase is a rotary motor, the smallest yet known. Understanding how this enzyme works will aid in identifying and characterizing other cellular motors. Ongoing studies in this laboratory are aimed at defining the molecular details of how the cooperative functioning of three catalytic sites in ATP hydrolysis or ATP synthesis is coupled to proton translocation through a proton pore. This appears to involve a rotor including the gamma-epsilon and 12 c subunits of the enzyme moving against a stator of the alpha3beta3deltab2 and a subunits in the F1F0 from escherichia coli. Crystallographic and electron microscopy approaches are being used to examine the structure of the enzyme at different positions in the rotation cycle. In humans, two of the subunits of the F1F0, subunits 6 and A6L, are encoded on mitochondrial (mt) DNA. Pathogenic mutants of mtDNA are being reported with increased frequency, including ones in subunits 6. Human F1F0 has been isolated from fibroblast cell lines for the first time in this laboratory. Studies are planned to examine the enzymatic properties and the assembly of F1F0 in cells of patients harboring subunit 6 mutations.

F/1F/O type ATPases are an important group of membrane-bound multisubunit enzymes involved in using ATP to generate a proton gradient, which can be used for substrate or ion transport. In mitochondria chloroplasts and in the plasma membrane of bacteria, the F/1F/O type ATPase also synthesizes ATP. We are studying the structure of the F/1F/O from Escherichia coli (ECF/1F/O) by a number of different approaches including cryoelectron microscopy, chemical cross-linking, protease digestion experiments, nuclear magnetic resonance studies, and fluorescence methods. We are also examining the conformational changes in ECF/1F/O that are involved in functioning of the enzyme complex. This aim is greatly aided by the fact that we can mutate subunits of the enzyme to include cysteine residues at defined positions in their sequences. These introduced cysteine residues are used to covalently attach reporter groups of conformational changes. In this way, kinetic analyses of conformational changes at various sites in the enzyme complex can be performed.

This award will support the participation of ten U.S. scientists in a U.S.-Japan Seminar on Gene Regulation and Expression in Mitochondrial Disease and the Aging Process. The seminar, which will be held December 7-12, 1992, in Honolulu, Hawaii, is being co-organized by Professor Roderick Capaldi, Institute of Molecular Biology, University of Oregon, and Dr. Takayuki Ozawa, Department of Biomedical Chemistry, Nagoya University. The seminar will focus on the latest developments in basic research on mitochondrial biogenesis, combining the interests of scientists who study the basic process of oxidative phosphorylation with those who study mitochondrial dysfunction in a wide variety of diseases and in the aging process. In addition to U.S. and Japanese scientists, the seminar will include researchers from Australia. This seminar follows a successful seminar in December 1987. The seminar topics are: structure and replication of mitochondrial DNA, regulation of mitochondrial molecular biology, biochemistry of mitochondrial enzyme complexes, bioenergetics and relationship to mitochondrial disease, nature of mitochondrial mutations associated with disease, mitochondria and the aging process, and treatment of mitochondrial diseases. The goal of the seminar is to encourage the sharing of expertise, which could lead to collaboration in this important area of research.

9317993 Dahlquist Partial funding of the hardware needed to perform pulsed field gradient experiments to enhance the use of nuclear magnetic resonance to study the solution properties of proteins and nucleic acids has been requested. The requested capability will greatly aid in 8 projects outlined in this proposal. These include investigations of the substrate binding site of galactose oxidase, the solution structure of the SYMBOL 101 \f "Symbol" subunit of the mitochondrial ATP synthase, the structure of an unusual DNA binding domain found in the project of the skn-1 gene of C. elegans, the structural basis of the interaction of two yeast transcription factors a1 and a2, the structural changes associated with mutations of the prosequence of the yeast carboxypeptidase and the folding pathway of the heterodimeric bacterial luciferase. s t g ? r StringFileInfo ^ 040904E4 ' CompanyName Microsoft Corporation : & FileDescription Windows Task Manager app9317993 Dahlquist Partial funding of the hardware needed to perform pulsed field gradient experiments to enhance the use of 4 5 K L o q $ $ $ D Y Y ? CG Times Symbol & Arial Tms Rmn Times New Roman Y 9 A A " h B e ~ < 1 Shauna Benson Deseree King, BIR

F1F0 type ATPases are found in mitochondria of animal and plant cells, in chloroplasts of plant cells, and in the plasma membrane of bacteria. They are responsible for more than 95% of the ATP production in cells and also function as an ATPase, using the energy derived from cleavage of ATP to generate a proton gradient, which is then used for substrate and ion transport. Defects in the mitochondrial F1F0 type ATPase can be the primary cause, and are often a secondary consequence, of several human diseases. The structure of this complicated enzyme, and its mechanisms of functioning -- particularly the way in which the conversion of chemical energy and chemiosmotic energy are interconverted -- are poorly understood. We are studying the structure of the F1F0 from Escherichia coli (ECF1F0) by a number of different approaches including cryoelectron microscopy, chemical cross linking, and nuclear magnetic resonance studies. Our immediate goal is to characterize the delta and b subunits structurally, and understand their arrangement in the intact F1F0 complex. We believe that these subunits form a scaffold (a stator) that fixes the alpha3beta3 domain to the F0 part of the complex, thereby allowing the rotation of a mobile element made up of the gamma and epsilon subunits. Our idea is that this mobile element links the three different catalytic sites, in turn, with a single proton channel in the F0 to maximize the efficiency of energy coupling.

Parkinson's disease is related to a deficiency of complex I bioenergetic activity that may be etiologic and likely contributes to increased oxidative stress. The protein chemical work described here addresses the question inherent to other parts of this Udall proposal, i.e., "As Complex I activity is clearly reduced in Parkinson's Disease, based on several studies, is this altered functioning caused by disrupted assembly and/or altered stability, AND/OR is there post-translational oxidative modification of the complex that progressively inhibits functioning? Project 3 will address three Aims. Specific Aim 1: Complete technique development and establish hot spots of oxidative modification of Complex I. While we have made considerable progress in identifying the sites of oxidative damage in Complex I by peroxynitrite, we need to fully identify sites of modification by hydroxyl radical reaction. Also, we wish to develop an antibody that can detect tryptophan oxidation to facilitate identification of this modification. Specific Aim 2: Further characterization of the phosphorylation/dephosphorylation of Complex I. We have established that three subunits of Complex I are phosphorylated and there is evidence that these modifications affect activity. Recent work has identified a rare genetic form of Parkinson's disease due to mutation of a mitochondrial protein kinase. Thus, altered phosphorylation/dephosphorylation may be a factor in the altered Complex I functioning which is evident in PD. We plan to identify the phosphorylation sites on Complex I by mass spectrometry and evaluate the functional significance of each. Specific Aim 3: Analysis of Complex I in PD brains and the cell lines created by protofection technology in Dr. Bennett's project. We have developed the tools for analyzing the levels of Complex I, its activity and the assembly state in small amounts of mitochondria. We will use these techniques to evaluate the properties of Complex I in PD brains and cell lines, and appropriate controls. These same samples will also be analyzed for oxidative, post-translational modifications that could account for altered functioning of Complex I.