Site-directed alkylation studies with LacY provide evidence for the alternating access model of transport.
In total, 59 single Cys-replacement mutants in helix VII and helix X of the lactose permease of Escherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles to complete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively low at the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cys substitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward the cytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing the central hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugar binding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. When considered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extend support for the alternating access model.
Department of Physiology, University of California, Los Angeles, California 90095, United States.
Xiaoxu
X
Jiang
Yiling
Y
Nie
H Ronald
HR
Kaback
D000478:Alkylation; D001692:Biological Transport; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary
D026901:Membrane Transport Proteins; C021499:lactose permease
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