Site-Directed Alkylation Studies with LacY Provide Evidence for the Alternating Access Model of Transport

摘要

In total, 59 single Cys-replacement mutants in helix VII and helix X of the lactose permease ofnEscherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles toncomplete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively lownat the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cysnsubstitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward thencytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing thencentral hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugarnbinding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. Whennconsidered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extendnsupport for the alternating access model.