Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites

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dc.contributor.authorWu, J.en
dc.contributor.authorFrillingos, S.en
dc.contributor.authorVoss, J.en
dc.contributor.authorKaback, H. R.en
dc.date.accessioned2015-11-24T19:07:18Z
dc.date.available2015-11-24T19:07:18Z
dc.identifier.issn0961-8368-
dc.identifier.urihttps://olympias.lib.uoi.gr/jspui/handle/123456789/20420
dc.rightsDefault Licence-
dc.subjectAmino Acid Sequenceen
dc.subjectAnilino Naphthalenesulfonates/pharmacologyen
dc.subjectBacterial Proteins/antagonists & inhibitors/*chemistry/genetics/metabolismen
dc.subjectBinding Sitesen
dc.subjectCoumarins/pharmacologyen
dc.subjectCysteine/chemistryen
dc.subjectElectron Spin Resonance Spectroscopyen
dc.subjectEscherichia coli/*enzymology/geneticsen
dc.subject*Escherichia coli Proteinsen
dc.subjectEthylmaleimide/pharmacologyen
dc.subjectIsopropyl Thiogalactoside/pharmacologyen
dc.subjectLactose/*metabolismen
dc.subjectLigandsen
dc.subjectMembrane Transport Modulatorsen
dc.subjectMembrane Transport Proteins/antagonists &en
dc.subjectinhibitors/*chemistry/genetics/metabolismen
dc.subjectMolecular Sequence Dataen
dc.subject*Monosaccharide Transport Proteinsen
dc.subjectMutagenesis, Site-Directeden
dc.subjectProtein Conformation/*drug effectsen
dc.subjectRecombinant Fusion Proteins/chemistry/metabolismen
dc.subjectSpectrometry, Fluorescenceen
dc.subjectSpin Labelsen
dc.subject*Symportersen
dc.subjectThiogalactosides/pharmacologyen
dc.subjectValine/chemistryen
dc.titleLigand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sitesen
heal.abstractBy 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)en
heal.accesscampus-
heal.fullTextAvailabilityTRUE-
heal.identifier.primary10.1002/pro.5560031214-
heal.identifier.secondaryhttp://www.ncbi.nlm.nih.gov/pubmed/7756985-
heal.identifier.secondaryhttp://onlinelibrary.wiley.com/store/10.1002/pro.5560031214/asset/5560031214_ftp.pdf?v=1&t=h0bz0dym&s=c073398db79b711de5def6836776be5ca2227f3f-
heal.journalNameProtein Scien
heal.journalTypepeer-reviewed-
heal.languageen-
heal.publicationDate1994-
heal.recordProviderΠανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικήςel
heal.typejournalArticle-
heal.type.elΆρθρο Περιοδικούel
heal.type.enJournal articleen

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