Synthesis of ATP during oxidative phosphorylation occurs on the F1Fo-ATP synthase enzyme and accounts of the bulk of ATP synthesis in living cells. ATP synthase is an extraordinary enzyme because it acts as a molecular motor the energy of the transmembrane proton gradient is coupled through subunit rotation to the synthesis of ATP in three asymmetric but interconverting catalytic sites. In reverse, ATP hydrolysis drives subunit rotation and proton pumping. The long-term goal of this research is to understand the mechanism of F1Fo-ATP synthase in as much molecular detail as possible. The E. coli enzyme will be used because of its many advantages, e.g. it is readily amenable to molecular biology/genetic manipulations, it may be rapidly obtained in high yield, and it may be reconstituted in liposomes with excellent ATP synthesis activity. Specific goals are (1) determination of catalytic sites occupancy and nucleotide binding parameters in F1Fo in presence of a proton gradient, during ATP synthesis; (2) identification of functional interactions between gamma/alpha and gamma/beta subunits, by mutagenesis of residues in gamma which face alpha and beta; (3) elucidation of protein movements generated at the catalytic alpha/beta subunit interface as ATP hydrolysis proceeds through formation and collapse of the transition state to the ADP ground- state; (4) characterization of ATP hydrolysis at low ATP concentrations, where 120 degrees Celsius stepping of the rotor is seen; and (5) genetic analysis of the stator stalk, starting from mutations in alpha and delta subunits shown previously in this laboratory to interrupt both F1 binding to Fo and energy coupling. ATP-driven pumps are very widely distributed in nature, and are involved in many disease states. Work to be done here will consequently have broad impact in biology and medicine.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025349-25
Application #
6525745
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Preusch, Peter C
Project Start
1978-09-01
Project End
2005-08-31
Budget Start
2002-09-01
Budget End
2003-08-31
Support Year
25
Fiscal Year
2002
Total Cost
$408,154
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of 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
Ahmad, Zulfiqar; Senior, Alan E (2004) Role of betaAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F(1)-ATPase. J Biol Chem 279:46057-64
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

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