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

被引:19
作者
Wu, JH
Hardy, D
Kaback, HR
机构
[1] Univ Calif Los Angeles, HHMI, MacDonald Res Labs 5 748, Dept Physiol, Los Angeles, CA 90095 USA
[2] Univ Calif Los Angeles, HHMI, Dept Microbiol & Mol Genet, Inst Mol Biol, Los Angeles, CA 90095 USA
关键词
D O I
10.1021/bi982288z
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The six N-terminal transmembrane helices (N-6) and the six C-terminal transmembrane helices (C-6) in lactose permease, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices V and VII, VIII, or X was studied by thiol-specific chemical cross-linking. The results demonstrate that Cys residues in the periplasmic half of helix V cross-link with Cys residues in the periplasmic half of helix VII, In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices V and VII. Moreover, Cys residues on one entire face of helix V cross-link with Cys residues on one 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 another toward the cytoplasmic side of the membrane. Furthermore, helices V and Vm are in close proximity throughout their lengths and do not tilt appreciably with respect to one another, and helices V and X are in close proximity at the cytoplasmic but not at the periplasmic face of the membrane.
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页码:2320 / 2325
页数:6
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