title: Chemical modification of mono-cysteine mutants allows a more global look at conformations of the epsilon subunit of the ATP synthase from Escherichia coli.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275-0376, USA.
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Ganti
authors.forename: Steven B
authors.initials: SB
authors.lastname: Vik
mesh_terms: 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
chemical_list: 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