An understanding of the molecular nature of membrane transport is an important objective of contemporary biological science. Previous work in this laboratory has established the ATPase in the plasma membrane of Neurospora crassa as an excellent model system for investigating membrane transport, and our present long-term goal is an understanding of the molecular mechanism by which this enzyme transduces the chemical energy of ATP hydrolysis into a transmembrane electrochemical protonic potential difference. In order to realize this goal, detailed information as to the molecular structure of the ATPase, its orientation in the membrane, and the dynamics of the ATPase molecule as it proceeds through the various stages of its catalytic cycle, will have to be obtained. The goals of this proposal are to begin to provide such information using predominantly chemical approaches. Specifically, we plan 1) to complete the purification and N-terminal amino acid sequencing of the H+-ATPase cyanogen bromide fragments, 2) to establish the positions in the 105,000 dalton polypeptide chain of several specific amino acid residues or sites already defined in the molecule, including the phosphorylatable aspartate, the site of action of the inhibitor, N- ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, the reactive thiols, the disulfide bridges, and a 7000 dalton fragment necessary for activity, 3) to define and establish the positions in the polypeptide chain of a variety of new sites in the molecule, including additional active site residues, exposed and inaccessible residues in several defined states of the molecule, residues exposed on the exocytoplasmic surface during the catalytic cycle, and several other sites as well, 4) to further investigate the events occurring in the catalytic cycle of the H+-ATPase, and 5) to measure several key intraenzymic distances in the ATPase molecule at several stages of its catalytic cycle using fluorescence energy transfer techniques. It is anticipated that what is learned will not only enhance our understanding of the molecular mechanism of the Neurospora plasma membrane H+-ATPase, but will also contribute to an understanding of membrane transport and membrane proteins in general.
