A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.
Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.
Jung
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Howard Hughes Medical Institute, Department of Physiology, University of California at Los Angeles 90024-1574.
Jung
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Kaback
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D003545:Cysteine; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000378:metabolism; D005453:Fluorescence; D005697:Galactosides / Q000494:pharmacology; D007700:Kinetics; D007785:Lactose / Q000494:pharmacology; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D008301:Maleimides / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D027981:Symporters; D014633:Valine
D029968:Escherichia coli Proteins; D005697:Galactosides; C050221:LacY protein, E coli; D008024:Ligands; D008081:Liposomes; D008301:Maleimides; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; C007816:beta-galactoside; C021499:lactose permease; D014633:Valine; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide