Ligand‐Induced conformational changes in the lactose permease of escherichia coli: Evidence for two binding sites

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right‐side‐out membrane vesicles containing Val 331 → Cys permease, lactose transport is inactivated by either N‐ethylmaleimide (NEM) or 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin (CPM). Remarkably, β,d‐galactopyranosyl 1‐thio‐β,d‐galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331 → Cys permease was then purified and studied in dodecyl‐β,d‐maltoside by site‐directed fluorescence spectroscopy. The reactivity of Val 331 → Cys permease with 2‐(4′‐maleimidylanilino)‐naphthalene‐6‐sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (