Structure-Function Studies of E. coli F1Fo-ATPase

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DESCRIPTION (provided by applicant): The mitochondrial F1-F0 ATP synthase catalyzes synthesis of the vast majority of ATP that is utilized by mammalian cells, in the culmination of an intricate process known as oxidative phosphorylation. It is a multisubunit, membrane-bound enzyme that is known to function with rotary motion of some of its subunits. Mutations found in several of its subunits are manifested clinically. Close relatives of the mitochondrial enzyme are found in chloroplasts and in some bacteria. Many of the recent insights into the structure and function of the ATP synthase have come from studies of the E. coli enzyme. This version of the enzyme contains eight different types of subunits. Alpha, beta, gamma, delta, and epsilon form FI, containing the sites of ATP synthesis. Subunits a, b and c form the membrane sector F0, containing the proton pathway. The movement of protons through F0 is thought to drive the rotation of gamma and epsilon subunits, relative to the alpha and beta subunits, which form the ATP catalytic sites. The studies proposed in this application focus on two of the subunits from the E. coli ATP synthase, epsilon and subunit a. The long term objectives of this project are to elucidate mechanisms of proton translocation and conformational coupling in the F1F0 ATP synthase. The focus of these studies is on the pathways of the protons that drive conformational and rotational movements of subunits in the ATP synthase, and on the conformational changes and binding sites of proteins involved in the transmission of mechanical energy to the sites of ATP synthesis. Four specific aims will be pursued. (A) Structure and dynamics of subunit a will be examined using cysteine-substitution mutagenesis, followed by disulfide formation and spin-labeling (B) Functional issues in subunit a will be addressed by mutagenesis, followed by assays of ATP synthesis. (C) Subunit interactions among F0 subunits will be investigated by engineering disulfide cross-linking. (D) Structural issues in the epsilon subunit that relate to its role in function during ATP synthesis and hydrolysis will be examined.