Apr 11, 2020

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

BioRxiv : the Preprint Server for Biology
Aurélie RothCédric Govaerts

Abstract

The Major Facilitator Superfamily (MFS) is the largest family of secondary active membrane transporters and is found in all domains of Life. Their transport cycle mechanism involves conformational changes between outward- and inward-open states. Yet, the kinetic details of MFS proteins have been largely missing. To investigate MFS transporters in real-time, we studied the multidrug efflux pump LmrP as a model system for antibiotic resistance development in bacteria. While it is well-known that the conformational cycle of LmrP is triggered by the protonation of a network of specific amino acids, the role of the transported substrate in these transitions is still puzzling. For a robust readout of the real-time structure of LmrP, we performed solution-based single-molecule Foerster resonance energy transfer (smFRET) using a confocal microscope with direct alternating donor/acceptor excitation and multiparameter (intensity, lifetime, anisotropy) detection. Lowering pH from 8 to 5 triggered an overall conformational transition, corroborating that detergent solubilization allows studying the LmrP transport cycle using smFRET. Using a newly developed linear 3-state photon distribution analysis (PDA) model, we show that the apo protein...Continue Reading

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