Stability and functionality of cysteine-less F(0)F1 ATP synthase from Escherichia coli

All 21 native cysteines in the Escherichia coli F(0)F1 ATP synthase were replaced by alanines. In isolated E. coli membranes, ATP-dependent proton pumping, turnover of ATP hydrolysis and steady-state transition state thermodynamic parameters of the cysteine-less enzyme were similar to wild-type. The cysteine-less enzyme was solubilized in n-octyl beta-D-glucopyranoside, purified by affinity chromatography, and reconstituted into pre-formed liposomes made from E. coli lipids. The properties of the reconstituted, purified enzyme were not significantly different from the membranous enzyme. These data demonstrate that cysteine-less F(0)F1 is biochemically stable and has functionality similar to wild-type.