Innovative Tools for the Structural and Functional Investigation of a Multidrug Efflux Pump from Pseudomonas Aeruginosa.

Efflux pumps are macromolecular assemblies that allow for proton-driven transport across both membranes in Gram-negative bacteria. In Pseudomonas aeruginosa, transport is made possible by the reversible assembly of a tripartite protein complex consisting of MexB, a membrane protein responsible for the active transport (energized by the proton motive force), MexA, a periplasmic protein whose putative role is to stabilize the whole complex and OprM an exit channel composed of a s-barrel inserted in the outer membrane and of a bundle of α-helices running along the periplasmic space.Such complexes are involved in the resistance against antibiotics, a field where new tools are needed for a better understanding of substrate transport. We have decided to investigate these pumps on two front lines.First, we have set up methodological developments for the design and production of a new set of synthetic scaffolds (dubbed α-reps for “artificial alpha repeat protein”) as membrane protein stabilizers and crystallization ortheses. Such interactants are selected through in vitro screening of the membrane proteins targets. We make use of amphipathic polymers (amphipols) to stabilize and immobilize the proteins onto solid support so that the library of possible interactants can be screened.As a complementary approach, we are working on the functional reconstitution of the pumps into proteoliposomes. Very recently we have designed a functional test for MexB. This original activity assay uses bacteriorhodopsin (BR), a light-activated proton pump, to generate a tunable, robust and reversible proton gradient. In this system, upon illumination with visible light, the photo-induced proton gradient created by the BR is shown to be coupled to the active transport of substrates through the pump. We are now working on the reconstitution of the whole efflux pump.