Tertiary contacts of helix V in the lactose permease determined by site-directed chemical cross-linking in situ.
The six N-terminal transmembrane helices (N6) and the six C-terminal transmembrane helices (C6) 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 VIII 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.
Wu
J
J
Department of Physiology, Howard Hughes Medical Institute, University of California, Los Angeles 90095-1662, USA.
Hardy
D
D
Kaback
H R
HR
D019943:Amino Acid Substitution / Q000235:genetics; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019897:Periplasm / Q000201:enzymology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D027981:Symporters
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D027981:Symporters; C021499:lactose permease; D003545:Cysteine