Site-directed Alkylation of LacY: Effect of the Proton Electrochemical Gradient

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H+ electrochemical gradient (Δμ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δμ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY.