The primary long-term objective of this study is to understand the mechanism by which cells (i.e. bacteria, mitochondria and chloroplasts) synthesize ATP. The system of choice for this study is the F1-Fo ATPase of E. coli. Of secondary interest is to learn about the various aspects of assembly of this multi-subunit, membrane-bound enzyme. This study proposes to focus on two subunits, which seem to be instrumental in two of the most interesting aspects of the enzyme. First is the alpha subunit, which seems to be involved in a proton channel through the membrane. This channel allows a proton gradient to drive net ATP synthesis. Second is the epsilon subunit, which is involved in the physical linkage of the membrane-bound subunits and the catalytic subunits, and in the regulation of the latter. The mechanism of any F1-Fo ATPase is relevant to (if not identical with) the mitochondrial enzyme, and hence, to the human condition, e.g. heart disease. Furthermore, this proton channel is probably related to the various ion pumps and channels which account for much of membrane biology. Specifically, this study aims to identify amino acid residues important or essential for the conduction of protons across the membrane. Recent evidence suggests that the carboxy-terminus of the alpha subunit is involved in this function. Individual amino acids in the E. coli protein, in particular those conserved among mitochondrial and chloroplast enzymes, will be replaced by other amino acids through a site-specific mutagenesis technique (cassette mutagenesis). The effect of mutations on growth properties of the cells, as well as the enzymatic properties of isolated membranes and proteins will be tested. The second specific aim involves mutagenesis of unc C, the gene for the epsilon subunit. A plasmid containing the entire gene will be subjected to in vitro mutagenesis and then used to transform cells unable to produce an epsilon subunit. Transformants will be screened for the inability to grow an succinate as the sole carbon source. Potentially, several types of mutations will arise: Those which diminish binding of F1 to the membrane but impair the functioning of the enzyme. Such studies intend to locate functions to specific domains of the epsilon subunit, and these domains can later be subjected to site-specific mutagenesis in order to probe more carefully structure-function relationships in this enzyme.
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