The long term objective of this study is to understand the mechanism by which cells (i.e., bacteria, mitochondria and chloroplasts) synthesize ATP, the most important metabolite in any organism. The system of choice for this study is the F1 FO-ATP synthase from Escherichia coli. The net synthesis of ATP is known to be coupled to the movement of protons across a membrane. The mechanism of any F1 F0-ATPase is thought to be closely related to that of the well-studied, mammalian mitochondrial enzymes, and therefore, such studies will be relevant to the human condition. In particular, many aspects of heart disease are likely to be related to the ability to make ATP and to utilize a transmembrane proton gradient. This study will focus on two subunits of the enzyme, which are involved in two of the most interesting aspects of its function. First is the alpha subunit, which makes a part of the proton channel through the enzyme. This channel allows a proton gradient to drive net ATP synthesis. Second is the epsilon subunit, which is necessary for the physical linkage between the membrane-bound subunits (F0) and the catalytic subunits (F1), and which functions as an intrinsic inhibitor. This study seeks to relate the structure of these two subunits to their function. In the case of the epsilon subunit, site-directed mutagenesis will be carried out in order to identify amino acid residues important binding for inhibition. It will be interesting to learn if all mutations are similarly defective in both binding and inhibition. In the case of the alpha subunit, several amino acid residues have already been identified as being involved in proton movement. Site-directed mutagenesis will continue to be applied in order to identify other amino acid residues that are important. Other approaches will serve to consolidate the information gained from mutagenesis. Topographical information will be gathered by introducing unique cysteine residues at various locations, and testing for the ability to be labelled from one side of the membrane. Finally, antibodies will be produced against native F0, by screening the lambda expression system. These can then be used to distinguish between local and global alterations in structure among the various mutants.
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