Lateral Diffusion and Association of Transmembrane Proteins Inside a Biomimetic Bilayer Mesophase

We have studied the properties of a sponge phase made of pentaethylene glycol monododecyl ether, a non-ionic surfactant, water (or alternatively a buffer), and a co-surfactant among a choice of four detergents widely used with membrane proteins. The phase was characterized by cross-polarized electron microscopy, diffusion of a fluorescent probe (fluorescence recovery after pattern phtobleaching), freeze fracture electron microscopy, and small angle x-ray and neutron scattering, and partial phase diagrams were created.This phase offers several advantages for membrane protein studies; the distance between unsupported bilayers can be tuned easily, the phase is non-viscous and hence easy to handle; it is isotropic and thus amenable to optical studies as well as for crystallization assays. Additionaly, we have demonstrated that several transmembrane proteins retain their structure and activity inside the phase.To demonstrate our approach we have studied the interaction between transmembrane proteins from a Pseudomonas aeruginosa efflux pump; MexA-MexB-OprM. By measuring their lateral diffusion we were able to determine the mode of interaction, the size of the protein complex and its stoichiometry. We have shown that MexA is embedded in the bilayer; that MexA and OprM do not interact laterally but form a complex if they are inserted in opposite bilayers and that the population of bound proteins is at its maximum for bilayers separated by a distance of ∼ 200 A, which is the expected periplasmic thickness for gram-negative bacteria.