Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides

Single cysteine-substitution mutants of KcsA, a K+ channel from Streptomyces lividans, were expressed in Escherichia coli, and inner membranes were isolated. The rate constants for the reactions of these cysteines with three maleimides of increasing hydrophobicity, 4-(N-maleimido)phenyltrimethylammonium, N-phenylmaleimide, and N-(l-pyrenyl)maleimide, were determined by back titration of the remaining cysteines with methoxypolyethylene glycol-2-pyridine disulfide (M-r 3,000) and quantitation of the fraction of gel-shifted KcsA as a function of reaction time. The patterns of the rate constants for the reactions of all three reagents with eight consecutive cysteines in the partially lipid-immersed amphipathic N-terminal tail helix were the same, with cysteines on the hydrophilic side of the helix reacting faster than Cys on the hydrophobic side. The results are consistent with the tail helix lying with its long axis in the lipid-water interface and with the orientation of the helix fluctuating around this axis. The patterns of the rate constants for the three reagents were similar to the pattern of the probabilities that the substituted cysteines were exposed to water, based on the sum of the free energies of transfer from water to octanol of all of the residues exposed to lipid in each orientation of the helix.