The molecular mechanism by which membrane transport proteins catalyze transmembrane solute movements remains an enigma despite decades of work on a variety of membrane transport molecules. Progress toward understanding this fundamental biological process has been hindered by a dearth of information as to the structures of membrane transport proteins at the molecular level of dimensions. This information can only be gained by electron and X-ray diffraction analyses of two-dimensional (2D) and three-dimensional (3D) crystals, but unfortunately, crystals of membrane transport proteins suitable for such analyses have been very difficult to obtain. The membrane transport protein of primary interest in this laboratory is the proton-translocating ATPase from the plasma membrane of Neurospora crassa. In the last few years, both 2D and 3D crystals of this enzyme suitable for structural analyses have been obtained, and a map of the structure of this transporter is emerging. Furthermore, a high-yield expression system for performing site-directed mutagenesis of the H+- ATPase molecule has also been developed. We are thus now in a position to carry out experiments that will begin to elucidate the molecular mechanism by which this transporter catalyzes ATP hydrolysis-driven, electrogenic proton translocation. In order to understand how the ATPase works, we must elucidate its molecular structure, define its molecular dynamics, identify its active site residues, define the path of proton flow through the enzyme from the cytoplasmic to the exocytoplasmic side of the membrane, and delineate the mechanism by which ATP hydrolysis drives an essentially unidirectional transmembrane proton flux. The experiments proposed in this application are designed to address these various aspects of the H+-ATPase molecular structure and mechanism. Specifically, we hope to l) extend the resolution of the currently available H+-ATPase structure map to 6 Angstroms or better in three dimensions and compare the structures of the ATPase in several distinct conformational states by cryoelectron crystallography of the 2D ATPase crystals, 2) improve current procedures for handling and growing the 3D H+-ATPase crystals and further extend the resolution of the ATPase structure map by X-ray diffraction analysis, and 3) utilize the site-directed mutagenesis system to define the location of the ATP hydrolytic site and other key sites in the H+-ATPase molecule by cryoelectron crystallography of gold-labeled single cysteine ATPase mutants, and define a variety of residues important for the function of the H+-ATPase molecule, including active site residues. All of the above approaches will then be used together in an attempt to elucidate the path of proton flow through the H+-ATPase molecule and the nature of the energy coupling process. It is anticipated that what is learned about the neurospora H+-ATPase will contribute to an understanding of the molecular mechanisms of the other ATPases in the aspartylphosphoryl-enzyme intermediate family and to an understanding of membrane-transport in general.
