Energy coupling is a fundamental process in biology that often involves the conversion of chemical energy to mechanical work. Efficient coupling is critical for active transport systems, yet the mechanics underlying this essential process are poorly understood. The yeast plasma membrane H+-ATPase is a P-type proton pump that couples ATP hydrolysis to proton transport. We have used this system to study coupling because it is highly suited to biochemical and genetic analyses that are essential for probing coupling. This proposal continues our detailed molecular probing of protein structure elements that contribute to coupling in the H+- ATPase. We plan to systematically probe by biochemical and genetic means the """"""""stalk' region, including its associated transmembrane segments, to better define their structural organization and role in coupling ATP hydrolysis to proton transport. The """"""""stalk"""""""" sector appears from low resolution structures of the closely related Ca2+-ATPase to physically link the cytoplasmic ATP hydrolysis and membrane-embedded proton transport domains. The importance of this region in coupling has emerged from genetic and biochemical studies in our lab, as well as others. The """"""""stalk"""""""" is believed to be comprised of 4-5 interacting alpha-helical elements which extend from transmembrane segments 1-5. Localized random mutagenesis will be used to generate mutations within the target region and potential pmal coupling mutants (partially uncoupled) will be selected on the basis of hygromycin B resistance and low pH sensitivity. The mutations will be genetically identified and mutant enzymes characterized for assembly and stability properties, the kinetics of ATP hydrolysis and proton transport, and the stoichiometry of H+ transported to ATP hydrolyzed. Scanning proline mutagenesis and targeted proteolysis will be used to explore backbone structure in the target region. Primary site mutations inducing prominent cellular and biochemical phenotypes will be used in suppressor studies to identify local and long-range protein structure interactions. Interactions between helical elements will be explored by targeted cysteine-directed crosslinking and a genetic dihybrid complementation system. Site-directed mutagenesis will be used to modify residues identified from the initial screening to be important to function and amino acid residues flanking important primary sites will be extensively modified by saturation mutagenesis to examine effects of localized structure on coupling. Finally, molecular modeling will be used as a visualization and prediction tool to model local regions of protein structure, as well as interactions between closely apposed protein structure elements. We expect that a mechanistic understanding of coupling by the H+-ATPase will be applicable to related P-type enzymes, other active transport systems, and diverse enzyme systems involved in energy coupling.
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