Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification

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dc.contributor.authorFrillingos, S.en
dc.contributor.authorKaback, H. R.en
dc.date.accessioned2015-11-24T18:56:26Z
dc.date.available2015-11-24T18:56:26Z
dc.identifier.issn0006-2960-
dc.identifier.urihttps://olympias.lib.uoi.gr/jspui/handle/123456789/19041
dc.rightsDefault Licence-
dc.subjectAmino Acid Sequenceen
dc.subjectBiotin/metabolismen
dc.subjectCarbohydrate Sequenceen
dc.subjectCell Membrane/enzymologyen
dc.subjectCysteineen
dc.subjectEscherichia coli/*enzymologyen
dc.subject*Escherichia coli Proteinsen
dc.subjectEthylmaleimide/*metabolism/pharmacologyen
dc.subjectKineticsen
dc.subjectMembrane Transport Proteins/*chemistry/metabolismen
dc.subjectMolecular Sequence Dataen
dc.subject*Monosaccharide Transport Proteinsen
dc.subjectMutagenesis, Site-Directeden
dc.subjectPlasmidsen
dc.subject*Point Mutationen
dc.subject*Protein Conformation/drug effectsen
dc.subjectRecombinant Proteins/chemistry/metabolismen
dc.subjectSulfhydryl Compoundsen
dc.subject*Symportersen
dc.subjectThiogalactosides/*pharmacologyen
dc.subjectValineen
dc.titleProbing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modificationen
heal.abstractBy using site-directed chemical labeling of lactose permease, conformational changes induced by ligand binding are observed in the native membrane of Escherichia coli. Membranes containing permease mutants with a single-Cys residue and a biotin-acceptor domain were labeled with radioactive N-ethylmaleimide (NEM) in the presence or absence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or a proton electrochemical gradient, followed by solubilization in n-dodecyl beta-D-maltopyranoside and adsorption to avidin. TDG-induced enhancement of the reactivity of membrane-embedded Val315-->Cys (helix X) permease is observed, while the reactivity of Val331-->Cys (helix X) permease is inhibited by ligand binding or imposition of a proton electrochemical gradient. In contrast, the reactivity of permease with a single native Cys residue at position 148 (helix V) is blocked by TDG, but unaffected by the proton electrochemical gradient. Furthermore, as shown with right-side-out and inside-out membrane vesicles, the accessibility of Cys148 to either NEM or impermeant methanethiosulfonate derivatives is comparable from both sides of the membrane. On the other hand, TDG protects Cys148 from alkylation more effectively in right-side-out vesicles (apparent KD = 20-50 microM) than inside-out vesicles (apparent KD ca. 1.0 microM). The findings provide strong support for the conclusion that the permease retains close to native conformation in n-dodecyl-beta-D-maltopyranoside. In addition, the results are consistent with the idea that lactose permease has two binding sites: one with higher affinity on the periplasmic surface of the membrane and another with lower affinity on the cytoplasmic surface.en
heal.accesscampus-
heal.fullTextAvailabilityTRUE-
heal.identifier.primary10.1021/bi952601m-
heal.identifier.secondaryhttp://www.ncbi.nlm.nih.gov/pubmed/8672426-
heal.identifier.secondaryhttp://pubs.acs.org/doi/pdfplus/10.1021/bi952601m-
heal.journalNameBiochemistryen
heal.journalTypepeer-reviewed-
heal.languageen-
heal.publicationDate1996-
heal.recordProviderΠανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικήςel
heal.typejournalArticle-
heal.type.elΆρθρο Περιοδικούel
heal.type.enJournal articleen

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