INTEGRATION OF F1 AND F0 ECTORS OF E. COLI PROTON-ATPASE

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Subunit "b" (also called "uncF" protein) is one of three subunits in the Fo-sector of E. coli proton-ATPase. Its amino acid sequence is known. Recent data implicates subunit b in specific binding of the F1-sector (the ATP synthesis/hydrolysis catalytic unit) to the Fo-sector (the proton-channel in the membrane). Taken together with predictions of the likely secondary and tertiary structure of subunit b, the evidence indicates that this subunit plays a central role in integration of the F1 and Fo sectors and in the coupling of ATP hydrolysis to H+ ion transport ("energy coupling"). In order to assess the importance of specific residues and domains of subunit b in F1-Fo integration, subunit b will be modified by a variety of techniques including chemical modification, limited proteolysis, localized mutagenesis. Functional correlations will be made by assaying the effects of the subunit b modifications on passive proton-transport through Fo specific F1-binding, ATP-driven proton-pumping and transmittal of proton-gradient-induced conformational changes to F1. Cross-linking experiments will be done to determine which residues or domains of subunit b bind to F1-subunits, and examination of partial revertants will be carried out as an alternative way of uncovering intersubunit interations between subunit b and other subunits of the proton-ATPase. The nature of "energy-coupling" in ion-transporting ATPases is a fundamental problem of biology. By focusing on subunit b we will deduce molecular insights into this process.