A functional his-tagged c subunit of the Escherichia coli F-type ATPase/synthase

The c subunit of the Escherichia coli F-0 has been tagged with a hexahistid ine motif at its C-terminus. The tagged subunit is capable of forming funct ional F-0 complexes that translocate protons in the absence of the F-1 comp lex. In the presence of F-1, the two sectors associate and display all bioc hemical activities of the wildtype enzyme: DCCD-inhibitable ATPase activity , ATP synthase activity, and ATP-dependent proton pumping. The enzyme can b e solubilized and purified as an intact complex under native conditions on immobilized-metal affinity chromatography (IMAC) resin. The purified comple x can be reincorporated into liposomes and demonstrates ATP-dependent proto n pumping activity. Hexahistine tags placed at the N-terminus, in contrast, were all inactive. These experiments demonstrate the feasibility of taggin g the c subunit for further studies of the F-0 and suggest an important rol e for the N-terminus of the c subunit in either assembly or function of the protein. (C) 2001 Academic Press.