Proximity relationships between helices I and XI or XII in the lactose permease of Escherichia coli determined by site-directed thiol cross-linking.
The lactose permease of Escherichia coli was expressed in two fragments (split permease), each with a Cys residue, and cross-linking was studied. Split permease with a discontinuity in either loop II/III (N2C10permease) or loop VI/VII (N6C6permease) was used. Proximity of multiple pairs of Cys residues in helices I and XI or XII was examined by using three homobifunctional thiol-specific cross-linking reagents of different lengths and flexibilities (6 A, rigid; 10 A, rigid; 16 A, flexible) or iodine. Cys residues in the periplasmic half of helix I cross-link to Cys residues in the periplasmic half of helix XI. In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices I and XI. Therefore, the periplasmic halves of helices I and XI are in close proximity, and the helices tilt away from each other towards the cytoplasmic face of the membrane. Cross-linking is also found with paired Cys residues near the middle of helices I and XII, but not with paired Cys residues near either end of the helices. Thus, helices I and XII are in close proximity only in the approximate middle of the membrane. Based on the findings, a modified helix packing model is proposed.
Wang
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Departments of Physiology and Microbiology and Molecular Genetics Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, 90095-1662, USA.
Kaback
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HR
D001693:Biological Transport, Active; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D008301:Maleimides; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013438:Sulfhydryl Compounds / Q000737:chemistry; D027981:Symporters
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008301:Maleimides; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; C075594:1,6-bismaleimidohexane; C021499:lactose permease; C011328:N,N'-4-phenylenedimaleimide; D007785:Lactose; D003545:Cysteine