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.
