Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ

The six N-terminal transmembrane helices (N-6) and the six C-terminal trans membrane helices (C-6) in lactose permease, each containing a single Cys re sidue, were coexpressed, and cross-linking was studied. The proximity of pa ired Cys residues in helices V and VII, VIII, or X was studied by thiol-spe cific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the peripla smic half of helix VII, In contrast, no cross-linking is evident with paire d Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cy s residues on one entire face of helix V cross-link with Cys residues on on e face of helix VIII, Finally, paired Cys residues at the cytoplasmic ends of helices V and X cross-link, but no cross-linking is observed when paired Cys residues are placed at the periplasmic ends of the two helices. Taken together, the results indicate that the periplasmic halves of helices V and VII are in close proximity and that the two helices tilt away from one ano ther toward the cytoplasmic side of the membrane. Furthermore, helices V an d Vm are in close proximity throughout their lengths and do not tilt apprec iably with respect to one another, and helices V and X are in close proximi ty at the cytoplasmic but not at the periplasmic face of the membrane.