A versatile Bilayer Phase for the Studies of Transmembrane Proteins’ Association

Abstract

The fluorescence recovery after pattern photobleaching (FRAPP) method was used to study the diffusion of membrane proteins, peptides, and lipids embedded into the bilayers of a biomimetic sponge phase.Lateral diffusion of membrane proteins is a useful tool for investigating the function, and association of these biopolymers. FRAPP results can also provide further information regarding their mobility, i.e. Brownian, accelerated versus slowed down, as well as their location: transmsmbrane, membrane or in between the bilayers of a sponge phase (L3).Using this L3 phase for membrane protein studies offers several advantages; the distance between unsupported bilayers is easily tunable; the phase is non-viscous and thus easy to handle; it is isotropic and therefore amenable to optical studies as well as for crystallization assays. In order to ascertain whether the phase is biologically relevant it was tested with several transmembrane proteins which retain their activity inside the phase.To illustrate our approach we will present the interaction between transmembrane proteins from a Pseudomonas aeruginosa efflux pump; MexA-MexB-OprM. By measuring their lateral diffusion we were able to deduce the mode of interaction, the size of the protein complex and its potential stoichiometry. Chiefly, we will illustrate 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.