Chemical modification of mono-cysteine mutants allows a more global look at conformations of the epsilon subunit of the ATP synthase from Escherichia coli.
The epsilon subunit of the ATP synthase from E. coli undergoes conformational changes while rotating through 360 degrees during catalysis. The conformation of epsilon was probed in the membrane-bound ATP synthase by reaction of mono-cysteine mutants with 3-N-maleimidyl-propionyl biocytin (MPB) under resting conditions, during ATP hydrolysis, and after inhibition by ADP-AlF(3). The relative extents of labeling were quantified after electrophoresis and blotting of the partially purified epsilon subunit. Residues from the N-terminal beta-sandwich domain showed a position-specific pattern of labeling, consistent with prior structural studies. Some residues near the epsilon-gamma interface showed changes up to two-fold if labeling occurred during ATP hydrolysis or after inhibition by ADP-AlF(3). In contrast, residues found in the C-terminal alpha-helices were all labeled to a moderate or high level with a pattern that was consistent with a partially opened helical hairpin. The results indicate that the two C-terminal alpha-helices do not adopt a fixed conformation under resting conditions, but rather exhibit intrinsic flexibility.
Ganti
Sangeeta
S
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA.
Vik
Steven B
SB
D000255:Adenosine Triphosphate / Q000378:metabolism; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D008301:Maleimides; D021122:Protein Subunits; D000255:Adenosine Triphosphate; C047272:3-(N-maleimidopropionyl)biocytin; D025243:Bacterial Proton-Translocating ATPases; D008239:Lysine; D003545:Cysteine