Transmembrane Protein Association in a Biomimetic Medium

Abstract

A plethora of membrane proteins studies have been performed on micellar systems containing detergent solubilised proteins. A major drawback of these systems is their rather poor mimicry of biological membranes. Therefore, development of new biologically relevant membrane systems is justified.We illustrate an original approach combining fluorescence recovery after fringe pattern photobleaching (FRAPP) with the use of an optically isotropic, extremely fluid phase of bilayers (the sponge phase). In this biologically relevant system, well suited for spectroscopic studies, the spacing between two adjacent bilayers and the thickness of the bilayers can be easily and very precisely tuned. So when the membranes are sufficiently separated, the only possible interactions occur between proteins embedded in the same bilayer, whereas when the membranes come together, interactions between proteins embedded in adjacent membranes may also occur.We present studies of interactions between proteins of the efflux pump of Pseudomonas aeruginosa: MexB (the rotor of the pump connecting the interior of the bacterium and the periplasm), OprM (a pipe connecting the periplasm to the outside of the bacterium) and perplasmic MexA. The mode of interaction, the size of protein complexes and their stoichiometry were determined. In particular we show that MexA and OprM interact only if they are incorporated in opposite bilayers. The population of this complex reaches its maximum when the bilayers are separated by a distance of about 200 A, which is the thickness of the Pseudomonas aeruginosa periplasm. The stoichiometry of the above complex will be presented as well as results describing Mexa-MexB and MexB-OprM associations.We will thus demonstrate the versatility of our system, which is well suited to study the associations of membrane proteins in a biologically relevant environment.