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dc.contributor.authorHealy, Jessica
dc.contributor.authorEkkerman, Silvia
dc.contributor.authorPliotas, Christos
dc.contributor.authorRichard, Morgiane
dc.contributor.authorBartlett, Wendy
dc.contributor.authorGrayer, Samuel C.
dc.contributor.authorMorris, Garrett M.
dc.contributor.authorMiller, Samantha
dc.contributor.authorBooth, Ian R.
dc.contributor.authorConway, Stuart J.
dc.contributor.authorRasmussen, Tim
dc.identifier.citationHealy , J , Ekkerman , S , Pliotas , C , Richard , M , Bartlett , W , Grayer , S C , Morris , G M , Miller , S , Booth , I R , Conway , S J & Rasmussen , T 2014 , ' Understanding the structural requirements for activators of the Kef bacterial potassium efflux system ' , Biochemistry , vol. 53 , no. 12 , pp. 1982-1992 .
dc.identifier.otherPURE: 122516046
dc.identifier.otherPURE UUID: 23bb24ad-e62a-47b3-9a54-661a2ac6d30b
dc.identifier.otherScopus: 84898059795
dc.identifier.otherORCID: /0000-0002-4309-4858/work/31524147
dc.descriptionThis work was supported by The Wellcome Trust (WT092552MA), The Leverhulme Trust (EM-2012-60\2), BBSRC SysMO (BB/F003455/1), a European Union Marie Curie ITN Award (NICHE; 289384)en
dc.description.abstractThe potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of 10–20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10–20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.
dc.rights© 2014 American Chemical Society. This is an article distributed in accordance with the terms of the Creative Commons Attribution (CC BY NC 4.0) license, which permits others to distribute, remix, adapt and build upon this work, for noncommercial use, provided the original work is properly cited.en
dc.subjectQR Microbiologyen
dc.titleUnderstanding the structural requirements for activators of the Kef bacterial potassium efflux systemen
dc.typeJournal articleen
dc.description.versionPublisher PDFen
dc.contributor.institutionUniversity of St Andrews.School of Chemistryen
dc.contributor.institutionUniversity of St Andrews.Biomedical Sciences Research Complexen
dc.description.statusPeer revieweden

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