The proton-ATPase of E. coli is responsible for generation of trans-membrane proton gradients and for ATP synthesis in oxidative phosphorylation. This proposal aims to study structure and catalytic mechanism of the F1-sector of the enzyme by focussing on the three subunits (Alpha, Beta and Gamma) which together form the minimal assembly (Alpha3Beta3Gamma) required for catalytic activity. Modification of Alpha, Beta and Gamma subunits will be carried out by localized mutagenesis, site-directed mutagenesis and affinity labeling in order to assess the importance of specific residues and domains of the subunits in structure and catalysis. Functional correlations will be established by studying the effects of modifications on negative cooperativity of nucleotide binding, total nucleotide binding, single-site catalysis, positive catalytic site cooperativity, and intersubunit conformational interaction. In order to further study intersubunit interactions, and with particular reference to contact regions possibly involved in catalytic cooperativity, partial revertants will be isolated and characterized and cross-linking experiments will be performed on mutant enzymes. Hybrid enzymes containing one or two copies of mutant Alpha (or Beta) subunits and two or one copies of Alpha (or Beta) will be constructed, and their catalytic properties established. Such experiments may provide a test of the cyclical three-site reaction schemes for F1 catalysis which have been proposed by our laboratory and by other workers. Catalytic properties of the normal Alpha3Beta3Gamma minimal unit will be established, and the effects of addition of pure Delta and Epsilon subunits singly and in combination will be assessed to show how these subunits affect the overall properties of F1. Isolated Alpha subunit will be crystallized. The nature of energy-coupling and catalysis in ion-transporting ATPases is a fundamental problem of biology. The long-term goal of this proposal is to deduce molecular insights into this problem.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025349-12
Application #
3272959
Study Section
Biochemistry Study Section (BIO)
Project Start
1978-09-01
Project End
1991-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
12
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Rochester
Department
Type
School of Medicine & Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Senior, Alan E; Muharemagic, Alma; Wilke-Mounts, Susan (2006) Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. Biochemistry 45:15893-902
Ahmad, Zulfiqar; Senior, Alan E (2006) Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride. FEBS Lett 580:517-20
Ahmad, Zulfiqar; Senior, Alan E (2005) Modulation of charge in the phosphate binding site of Escherichia coli ATP synthase. J Biol Chem 280:27981-9
Ahmad, Zulfiqar; Senior, Alan E (2005) Identification of phosphate binding residues of Escherichia coli ATP synthase. J Bioenerg Biomembr 37:437-40
Wilkens, Stephan; Borchardt, Dan; Weber, Joachim et al. (2005) Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy. Biochemistry 44:11786-94
Ahmad, Zulfiqar; Senior, Alan E (2005) Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding. FEBS Lett 579:523-8
Weber, Joachim; Senior, Alan E (2004) Fluorescent probes applied to catalytic cooperativity in ATP synthase. Methods Enzymol 380:132-52
Weber, Joachim; Wilke-Mounts, Susan; Nadanaciva, Sashi et al. (2004) Quantitative determination of direct binding of b subunit to F1 in Escherichia coli F1F0-ATP synthase. J Biol Chem 279:11253-8
Weber, Joachim; Muharemagic, Alma; Wilke-Mounts, Susan et al. (2004) Analysis of sequence determinants of F1Fo-ATP synthase in the N-terminal region of alpha subunit for binding of delta subunit. J Biol Chem 279:25673-9
Senior, Alan E; Weber, Joachim (2004) Happy motoring with ATP synthase. Nat Struct Mol Biol 11:110-2

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