The synthesis of ATP during oxidative phosphorylation is catalyzed by a reversible H+-translocating ATPase. The enzyme has an ATP hydrolyzing sector (F1) and a proton-translocating sector (FO), which can be disassociated from each other and studied as individual entities. We are styding the FO-sector of the H+-ATPase in Escherichia coli, which is composed of 3 types of subunits. The long-term goal is to define in chemical detail how the FO moiety translocates protons across the membrane, and how proton-flux is coupled to ATP synthesis in F1. The research proposed will provide much needed chemical information on the constitutents of the H+-conducting pathway through FO, and on the functional interaction between FO and F1. Detailed chemical information on the protein components will be required before detailed mechanistic models can be proposed and tested. We will analyze E. coli mutants in FO to determine the functional role of each subunit. We will assay for loss of the following functions: (1) FO mediated H+-translocation, (2) Binding of F1 to FO (3) coupling of H+-translocation from FO to ATP synthesis in F1. The mutants will be further analyzed to determine the site of mutation so that specific amino acid residues can be related to function. This will be done by DNA sequencing. We will also test the role of specific amino acids by site-specific mutagenesis. Information on the organization and structure of subunit in FO will be obtained by chemical approaches (including cross-linking, chemical modification, limited proteolysis, and peptide directed antibodies). This information will be related to folding models predicted from the primary amino sequence. The information provided by these studies should contribute to elucidation of the mechanism of oxidative phosphorylation. Study of this membrane enzyme, which may have an unprecedented structural complexity, should also contribute to our general knowledge of membrane structure. Study of the FO sector as an H+-transporter should increase our presently vague knowledge of membrane transporters. A detailed understanding of membrane transport carriers and pumps may ultimately prove important to many areas of medical science, extending from renal and cardiac physiology to cancer.
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