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Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence

Basting, Daniel; Lorch, Mark; Lehner, Ines; Glaubitz, Clemens

Authors

Daniel Basting

Professor Mark Lorch M.Lorch@hull.ac.uk
Professor of Public Engagement and Science Communication. Interim Head of Department for Chemistry, Biochemistry and Chemical Engineering

Ines Lehner

Clemens Glaubitz



Abstract

Efflux pumps of the small multidrug resistance family bind cationic, lipophilic antibiotics and transport them across the membrane in exchange for protons. The transport cycle must involve various conformational states of the protein needed for substrate binding, translocation, and release. A fluorescent substrate will therefore experience a significant change of environment while being transported, which influences its fluorescence properties. Thus the substrate itself can report intermediate states that form during the transport cycle. We show the existence of such a substrate-transporter complex for the EmrE homologMycobacterium tuberculosisTBsmr and its substrate ethidium bromide. The pH gradient needed for antiport has been generated by co-reconstituting TBsmr with bacteriorhodopsin. Sample illumination generates a ΔpH, which results in enhanced ethidium fluorescence intensity, which is abolished when ΔpH or ΔΨ is collapsed or when the essential residue Glu-13 in TBsmr is exchanged with Ala. This observation shows the formation of a pH-dependent, transient substrate-protein complex between binding and release of ethidium. We have further characterized this state by determining theKd, by inhibiting ethidium transport through titration with nonfluorescent substrate and by fluorescence anisotropy measurements. Our findings support a model with a single occluded intermediate state in which the substrate is highly immobile.—Basting, D., Lorch, M., Lehner, I., Glaubitz, C. Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence.

Journal Article Type Article
Publication Date 2008-02
Journal FASEB Journal
Print ISSN 0892-6638
Electronic ISSN 1530-6860
Publisher Federation of American Society of Experimental Biology
Peer Reviewed Peer Reviewed
Volume 22
Issue 2
Pages 365-373
APA6 Citation Basting, D., Lorch, M., Lehner, I., & Glaubitz, C. (2008). Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence. FASEB Journal, 22(2), 365-373. https://doi.org/10.1096/fj.07-9162com
DOI https://doi.org/10.1096/fj.07-9162com
Keywords Biotechnology; Genetics; Biochemistry; Molecular Biology
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