The F-type ATP synthases/ATPases are highly conserved multisubunit enzymes responsible for coupling energy transduction from covalent bond to electrochemical gradient. The enzyme is comprised of two major sectors: F1, a peripheral membrane ATPase complex, and Fo, an integral membrane proton channel. The F1 has been extensively analyzed, but inforamtion of the Fo has lagged behind, primarily due to complications of working with membrane proteins. The principle subunit of the Fo, the proteolipid, will be modified with a his6-tag by site-directed mutagenesis for the following purposes. First, to characterize by differential electrophoresis alternate forms of the Fo that occur under different growth conditions. Second, the modification will be used to improve current protocols for purification of Fo. Purified Fo will be reconstituted into vesicles and/or planar membranes and analyzed for proton conductance behavior. The data from these experiments will reveal mechanisms for regulating the coupling ratio and efficiency of the F-ATPase under different physiological conditions.
