Asymmetric Proteoliposomes - Striking a New Path in the World of Model Membranes

Nearly all natural lipid membranes show a lipid asymmetry between the inner and the outer leaflet and cells spend a substantial amount of energy on establishing and maintaining this asymmetry. This implies important biological functions of these specific asymmetries, but up to now, only little is known about this, because virtually all membrane protein studies have been performed in symmetrical model membranes. Recently, a number of protocols has been established to produce asymmetric liposomes that should allow for finally tackling this problem. We contributed two assays, to prepare phosphatidylglycerol (PG)-outside vesicles using PG-cyclodextrin complexes [1] and phosphatidylserine (PS)-inside ones by enzymatic treatment with a PS decarboxylase [2]. However, application of these protocols to proteoliposomes poses additional challenges: protein reconstitution may enhance lipid flip-flop and establishing asymmetry may destabilize the embedded protein. We think that the most important requirement for stable asymmetry is an optimal lipid packing around the membrane protein. To this end, we optimized the acceptor liposome composition of our recent PG asymmetry protocol [1] to better mimic the natural lipid environment of a bacterial membrane protein. We reduced phosphatidylcholine to 40 mol% needed for stable liposomes, replacing it by phosphatidylethanolamine and 2.5 mol% cardiolipin. Finally, we reconstituted a bacterial membrane protein before rendering the proteoliposome asymmetrical. We were able to establish conditions that allow for a stable, PG-outside asymmetry for more than 10 days while keeping the protein fully functional. This protocol opens an avenue to finally study asymmetry effects on functional parameters of a membrane protein in detail. [1] Markones et. al. Langmuir 2018 [2] Drechsler et al. BJ in press