Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.
By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
J
J
Department of Physiology, Howard Hughes Medical Institute, University of California at Los Angeles 90024-1662, USA.
Frillingos
S
S
Voss
J
J
Kaback
H R
HR
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003374:Coumarins / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism*; D008024:Ligands; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D027981:Symporters; D013862:Thiogalactosides / Q000494:pharmacology; D014633:Valine / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D003374:Coumarins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D013113:Spin Labels; D027981:Symporters; D013862:Thiogalactosides; D007544:Isopropyl Thiogalactoside; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C030860:N-(4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl)maleimide; C031803:thiodigalactoside; C021499:lactose permease; D014633:Valine; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide