F1F0-ATP synthase is responsible for the bulk of ATP synthesis in most organisms. It uses a transmembrane proton gradient to synthesize ATP from ADP and Pi, and it hydrolyses ATP to transport protons across the membrane. Both functions are tightly coupled by a unique mechanism, subunit rotation, making ATP synthase a very efficient rotary nanomotor. The broad, long-term goal of this research is to understand the mechanism of ATP synthase in molecular detail.
The Specific Aims focus on the connection between substrate binding/turnover in the three catalytic sites and subunit rotation, trying to exploit the available structural information to the fullest.
The Specific Aims are: (1) Identification of the high-affinity site, which is responsible for catalysis, in the x-ray structure. Fluorescence resonance energy transfer between tryptophan residues in the rotor and nucleotide analogs will be used to achieve this aim. (2) Investigation of the functional significance of subunit/subunit contacts specific to one of the three catalytic Beta/Alpha interfaces. The functional consequences of (a) preventing these contacts and (b) making them permanent by crosslinking will be tested. (3) Kinetic analysis of nucleotide binding to the catalytic site. ATP binding contributes to driving subunit rotation; the sequence of events leading from the initial contact to closure of the site will be analyzed using tryptophan fluorescence and rapid kinetics. (4) Analysis of the role of the C-terminal domain of Beta in driving rotation. A loop in this domain is generally regarded as instrumental for coupling catalysis to rotation. This hypothesis will be tested by reducing the size of the loop and measurement of the rotational torque produced by ATP hydrolysis. (5) Investigation of the contribution of the Alpha subunit in driving subunit rotation. The role of a loop in the C-terminus of Alpha in transmitting conformational changes between the catalytic sites and the rotor will be analyzed, again using deletions and torque measurements. ATP synthase from E. coli will be used as model in these studies. However, the basic mechanism of all ATP synthases is the same, independent of the source. Mutations in human mitochondrial ATP synthase and related ATP-driven pumps are responsible for a number of diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM071462-05
Application #
7497953
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Anderson, Vernon
Project Start
2004-09-01
Project End
2011-08-31
Budget Start
2008-09-01
Budget End
2011-08-31
Support Year
5
Fiscal Year
2008
Total Cost
$219,980
Indirect Cost
Name
Texas Tech University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041367053
City
Lubbock
State
TX
Country
United States
Zip Code
79409
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Mnatsakanyan, Nelli; Hook, Jonathon A; Quisenberry, Leah et al. (2009) ATP synthase with its gamma subunit reduced to the N-terminal helix can still catalyze ATP synthesis. J Biol Chem 284:26519-25
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De Angeli, Alexis; Moran, Oscar; Wege, Stefanie et al. (2009) ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles. J Biol Chem 284:26526-32
Mao, Hui Z; Abraham, Christopher G; Krishnakumar, Arathianand M et al. (2008) A functionally important hydrogen-bonding network at the betaDP/alphaDP interface of ATP synthase. J Biol Chem 283:24781-8
Weber, Joachim (2007) ATP synthase--the structure of the stator stalk. Trends Biochem Sci 32:53-6
Mao, Hui Z; Weber, Joachim (2007) Identification of the betaTP site in the x-ray structure of F1-ATPase as the high-affinity catalytic site. Proc Natl Acad Sci U S A 104:18478-83
Mao, Hui Z; Gray, Wesley D; Weber, Joachim (2006) Does F1-ATPase have a catalytic site that preferentially binds MgADP? FEBS Lett 580:4131-5
Weber, Joachim (2006) ATP synthase: subunit-subunit interactions in the stator stalk. Biochim Biophys Acta 1757:1162-70