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.
Kuo
P H
PH
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville 22906-0011, USA.
Ketchum
C J
CJ
Nakamoto
R K
RK
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001693:Biological Transport, Active; D003545:Cysteine / Q000737:chemistry; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D008081:Liposomes; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D012995:Solubility; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D001426:Bacterial Proteins; D003902:Detergents; D008081:Liposomes; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases; D003545:Cysteine