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Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence
Author(s) -
Basting Daniel,
Lorch Mark,
Lehner Ines,
Glaubitz Clemens
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.07-9162com
Subject(s) - ethidium bromide , chemistry , efflux , substrate (aquarium) , fluorescence anisotropy , antiporter , fluorescence , biophysics , membrane , biochemistry , dna , biology , ecology , physics , quantum mechanics
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 homolog Mycobacterium tuberculosis TBsmr and its substrate ethidium bromide. The pH gradient needed for antiport has been generated by co‐reconstituting TBsmr with bacteriorhodopsin. Sample illumination generates a ∆pH, which results in enhanced ethidium fluorescence intensity, which is abolished when ∆pH 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 substrateprotein complex between binding and release of ethidium. We have further characterized this state by determining the K d , 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. FASEB J. 22, 365–373 (2008)