Transport cycle intermediate in small multidrug resistance protein is revealed by substrate fluorescence.
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 DeltapH, which results in enhanced ethidium fluorescence intensity, which is abolished when DeltapH or DeltaPsi 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 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
Daniel
D
Intitut für Biophysikalische Chemie, J. W. Goethe Universität, Max von Laue Str. 9, 60438 Frankfurt, Germany.
Lorch
Mark
M
Lehner
Ines
I
Glaubitz
Clemens
C
D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D004351:Drug Resistance; D004996:Ethidium / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D011522:Protons; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics
D017920:Antiporters; D008055:Lipids; D008081:Liposomes; D011522:Protons; D004996:Ethidium