In vitro folding of a membrane protein: effect of denaturation and renaturation on substrate binding by the lactose permease of Escherichia coli.
Site-directed mutagenesis and site-directed fluorescence spectroscopy demonstrate that Cys148 interacts hydrophobically with the galactosyl moiety of substrates of the lactose permease of Escherichia coli. By taking advantage of the finding that labelling of single-Cys148 permease with the thiol-specific fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) is blocked specifically by substrates of the permease, it is demonstrated that the high-affinity ligand beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) stabilizes solubilized, purified permease against heat denaturation. Furthermore, TDG protection against MIANS labelling of single-Cys148 permease is abolished by guanidinium hydrochloride. After dialysis of the denaturant, TDG protection against MIANS labelling is recovered, indicating that the permease has been refolded. The conclusion is confirmed and extended by studying site-directed fluorescence of purified single-Cys331 permease, where the emission spectrum of the MIANS-labelled protein is differentially altered by low or high concentrations of TDG. The results demonstrate that both low- and high-affinity binding, as well as ligand-induced conformational changes in the permease, can be denatured reversibly in vitro.
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Howard Hughes Medical Institute, Department of Microbiology, University of California, Los Angeles 90095-1662, USA.
Kaback
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D019943:Amino Acid Substitution / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006358:Hot Temperature; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D027981:Symporters; D013862:Thiogalactosides / Q000494:pharmacology
D003902:Detergents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; C031803:thiodigalactoside; C021499:lactose permease; D003545:Cysteine