The overall aim of the research described is to establish the molecular mechanism of ATP hydrolysis by F/1-ATPases. The studies will focus on the F/1-ATPase from the thermophilic Bacillus PS3 (TF/1) and its alpha3beta3gamma subcomplex.
The specific aims are the following: 1. To identify amino acid residues participating in propagation of conformation signals from one catalytic site to another, site directed mutants of strategically located amino acid residues will be prepared and submitted to rigorous kinetic analyses. Of particular interest is identification of amino acid residues at the alpha/beta interface near non-catalytic sites that participate in positive catalytic cooperativity. 2. Wild-type and mutant alpha3beta3gamma subcomplexes of TF/1 will be photoinactivated with [3H]octylquinaldinium under conditions that the reagent stimulates ATPase activity in the absence of light.
The aim of this project is to locate the region of TF/1 that interacts with the reagent during the stimulatory process. 3. To assess the catalytic importance of the hydrogen bond between the beta-carboxylate of betaGlu/201 and the hydroxyl of betaThr/165 in the open form of catalytic sites in the alpha3beta3gamma subcomplex of TF/1. This will be accomplished by detailed characterization of site directed mutants of the subcomplex with in which betaGlu/201 is substituted with several amino acids. 4. To determine whether two or three catalytic sites are in closed conformations when three catalytic sites are saturated with MgATP, strategically placed pairs of cysteines have been introduced into the alpha3beta3gamma mutant subcomplex. The rate of oxidation of the triple mutants will be correlated with Mg[3H]ATP or Mg[3H]ADP sequestered by catalytic sites. 5. The -CCXXCC- motif will be introduced into alpha-helical segments of the gamma subunit to provide covalent binding sites for FlAsH, a 4', 5'- bis arsenical derivative of fluorescein. This will provide a probe for single molecule confocal fluorescence spectroscopy. This technique will be used to correlate rotation of the gamma subunit with ATP concentration and to monitor the effects of mutations on rotation.
