Speaker
Description
Plasma membranes are intrinsically asymmetric, with the two leaflets differing in lipid composition. This compositional asymmetry alters lateral pressure profiles, curvature, and hydrophobic thickness, which can modulate the function of embedded membrane proteins. To elucidate how membrane asymmetry couples to membrane protein function, our lab reconstitutes proteins into compositionally well-defined mimics of plasma membranes. We focus on the outer membrane phospholipase A (OmpLA), an integral membrane lipase from the outer membrane of Gram-negative bacteria. Asymmetric large unilamellar proteoliposomes (aPLUVs; size: ~ 100 nm) containing OmpLA were generated via cyclodextrin-mediated lipid exchange using mixtures of monounsaturated phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylserine. Membrane structure and thermotropic behavior were characterized by combined small-angle X-ray and neutron scattering, together with differential scanning calorimetry. To probe the functional consequences of asymmetry, we monitored OmpLA dimerization in asymmetric bilayers as a readout of its enzymatic activation. For this, we employed an inactive OmpLA variant site-specifically labeled with AF488 or AF647 to enable Förster resonance energy transfer (FRET), and performed single-molecule FRET measurements on a custom-built confocal microscope. Our results provide first insights into how lipid bilayer asymmetry modulates OmpLA dimerization and activity, highlighting a direct link between the asymmetric physical properties of the membrane and the functional state of an integral membrane enzyme.