title: Tilting of helix I and ligand-induced changes in the lactose permease determined by site-directed chemical cross-linking in situ.
abstract: The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of lactose permease, each with a single Cys residue, were co-expressed, and cross-linking was studied. The proximity of paired Cys residues in helices I (positions 11, 14, 15, 18, 25, 28, 29, or 32) and VII (positions 227, 231, 232, 234, 235, 238, 239, 241, 242, 245, or 246) was studied by using homobifunctional thiol-specific chemical linkers of different lengths and chemical properties. The results demonstrate that Cys residues on one face of the periplasmic half of helix I (positions 32, 29, 28, or 25) cross-link to Cys residues on one face of the periplasmic half of helix VII (242 or 245). In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices I (positions 11, 14, 15, or 18) and VII (positions 227, 230, 231, 232, 234, 235, 238, or 239). The results indicate that helices I and VII are in close proximity only at their periplasmic halves. Ligand binding decreases cross-linking efficiency of the Cys pair 28/245 or 25/242 with N, N'-o-phenylenedimaleimide (rigid 6 A) and increases efficiency with N,N'-p-phenylenedimaleimide (rigid 10 A) or 1,6-bismaleimidohexane (flexible 16 A), indicating that the inter-thiol distance is about 6 A in the absence of ligand and that ligand binding increases the distance up to 10 A. The inter-thiol distance for Cys pairs 29/245 or 32/245 is less than 6 A in the absence of ligand, and in the presence of ligand, distance increases to between 6 and 10 A. Taken together, the results indicate that ligand binding induces a translational or scissors-like rigid body movement of helix I and/or VII at the periplasmic interface between the helices.
authors.affiliation: Howard Hughes Medical Institute, Molecular Biology Institute, University of California, Los Angeles 90095-1662, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wu
authors.forename: D
authors.initials: D
authors.lastname: Hardy
authors.forename: H R
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D008024:Ligands; D008301:Maleimides / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D027981:Symporters; D013862:Thiogalactosides / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D008301:Maleimides; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; D013862:Thiogalactosides; C075594:1,6-bismaleimidohexane; C031803:thiodigalactoside; C021499:lactose permease; C011328:N,N'-4-phenylenedimaleimide; D003545:Cysteine; C023860:N,N'-2-phenylenedimaleimide