The principal goal of this project is to determine mechanistic features of the yeast Saccharomyces cerevisiae mitochondrial ATP synthase. This work is particularly relevant to cardiac tissue which is almost completely dependent on the mitochondrial ATP synthase for aerobic synthesis of ATP. An understanding of the structure and function of the ATP synthase is essential before mitochondrial myopathies and pathologies that alter oxidative-phosphorylation can be completely understood and treated. This study will reveal important basic features of the ATP synthase and, since the yeast enzyme is highly homologous to the human enzyme, it is also important for understanding mitochondrial myopathies and pathologies. The ATP synthase is composed of two distinct multimeric portions, the Fl and the Fo. The Fl contains the catalytic site while the Fo is thought to act as a proton pore and acts to deliver protons to F1 or to an intermediate subunit. The oligomycin sensitivity conforming protein (OSCP) is a prime candidate to act as such an intermediate subunit. OSCP is essential for effective binding of Fl to Fo, and as an intermediate subunit, may be involved in either proton translocation or energy transduction. The overall objective of this project is to determine the structure and function of OSCP.
The specific aims are: 1) to determine the thermodynamic binding constants of OSCP with Fl and Fo, 2) to delineate the functional domains of OSCP, 3) to determine the primary structural requirements of OSCP, 4) to determine the role of OSCP in energy transduction or proton translocation, 5) and to determine the crystal structure of OSCP. Mature yeast OSCP has been successfully expressed in E. coli., radiolabeled with [35S], and purified to homogeneity. Thermodynamic binding constants will be determined by equilibrium binding studies. The functional domains of OSCP will be determined by the combined use of specific deletion mutants and by inhibition of binding by antibodies generated against specific regions of OSCP. The role of OSCP will be determined by the isolation and biochemical analysis of mutants in OSCP in the ATP synthase will be determined by the isolation and biochemical analysis of mutants in OSCP. Three classes of mutants will be studied; dominant negative, cold sensitive, and non-conditional. A selection scheme has been developed to identify mutants in each class. Mutations can be considered as specific modifiers of OSCP and the determination of the biochemical defect in OSCP will provide evidence on the role of OSCP in ATP synthesis. The genetic analysis of the mutants will provide information on primary structural requirements of OSCP and pin point functional domains. To obtain an understanding at the molecular level of the effect of the mutations, in collaboration with Dr. Gregory Petsko, the crystal structure of OSCP will be solved. With the imminent solution of the crystal structure of mammalian Fl, the crystal structure of yeast Fl can be modeled and used to help determine the molecular interactions of OSCP with F1.
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