In vitro reconstitution of lipid‐dependent membrane protein topological switching (785.1)

Abstract

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. Systematic alteration of lipid membrane composition uncovered a role for lipid‐protein interactions in determining dynamic post‐assembly changes in and initial topogenesis of membrane proteins. Using an in vitro proteoliposome system in which lipid composition can be systematically controlled not only before (liposomes) but also after (fliposomes) reconstitution, we have determined the minimum and sufficient requirements for lactose permease (LacY) to flip between topologically distinct states. Changes in phosphatidylethanolamine levels after final protein folding resulted in re‐orientation of the original conformer mixture, establishing a dynamic reversibility in topology without molecular chaperone or translocon involvement. Using Förster resonance energy transfer approaches, we further investigated the kinetics of lipid exchange and LacY flipping. The rates of protein flipping in both directions were on a second scale and occurred almost instantaneously as the lipid composition of the proteoliposomes changed. These observations demonstrate a potential thermodynamically driven lipid‐dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within cells independent of other cellular factors.Grant Funding Source: Supported by NIH grant GM R37 20478