Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites
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peer-reviewed
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Protein Sci
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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)
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Λέξεις-κλειδιά
Amino Acid Sequence, Anilino Naphthalenesulfonates/pharmacology, Bacterial Proteins/antagonists & inhibitors/*chemistry/genetics/metabolism, Binding Sites, Coumarins/pharmacology, Cysteine/chemistry, Electron Spin Resonance Spectroscopy, Escherichia coli/*enzymology/genetics, *Escherichia coli Proteins, Ethylmaleimide/pharmacology, Isopropyl Thiogalactoside/pharmacology, Lactose/*metabolism, Ligands, Membrane Transport Modulators, Membrane Transport Proteins/antagonists &, inhibitors/*chemistry/genetics/metabolism, Molecular Sequence Data, *Monosaccharide Transport Proteins, Mutagenesis, Site-Directed, Protein Conformation/*drug effects, Recombinant Fusion Proteins/chemistry/metabolism, Spectrometry, Fluorescence, Spin Labels, *Symporters, Thiogalactosides/pharmacology, Valine/chemistry
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http://www.ncbi.nlm.nih.gov/pubmed/7756985
http://onlinelibrary.wiley.com/store/10.1002/pro.5560031214/asset/5560031214_ftp.pdf?v=1&t=h0bz0dym&s=c073398db79b711de5def6836776be5ca2227f3f
http://onlinelibrary.wiley.com/store/10.1002/pro.5560031214/asset/5560031214_ftp.pdf?v=1&t=h0bz0dym&s=c073398db79b711de5def6836776be5ca2227f3f
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en
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Γενική Περιγραφή / Σχόλια
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Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής