Chemical modification of mono-cysteine mutants allows a more global look at conformations of the ε subunit of the ATP synthase from Escherichia coli

Abstract

The ε subunit of the ATP synthase from E. coli undergoes conformational changes while rotating through 360° during catalysis. The conformation of ε 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-AlF₃. The relative extents of labeling were quantified after electrophoresis and blotting of the partially purified ε subunit. Residues from the N-terminal β-sandwich domain showed a position-specific pattern of labeling, consistent with prior structural studies. Some residues near the ε-γ interface showed changes up to two-fold if labeling occurred during ATP hydrolysis or after inhibition by ADP-AlF₃. In contrast, residues found in the C-terminal α-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 α-helices do not adopt a fixed conformation under resting conditions, but rather exhibit intrinsic flexibility.