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

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

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.

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

• Journal Article Type: Article
• Acceptance Date: Aug 16, 2007
• Online Publication Date: Sep 14, 2007
• Publication Date: 2008-02
• Journal: FASEB Journal
• Print ISSN: 0892-6638
• Electronic ISSN: 1530-6860
• Publisher: Federation of American Society of Experimental Biology (FASEB)
• Peer Reviewed: Peer Reviewed
• Volume: 22
• Issue: 2
• Pages: 365-373
• DOI: https://doi.org/10.1096/fj.07-9162com
• Keywords: Biotechnology; Genetics; Biochemistry; Molecular Biology
• Public URL: https://hull-repository.worktribe.com/output/409577