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Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes \textit{in vitro}, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), displayed slow spontaneous flip-flop at 37 oC (half-time, t_{1/2} = 140 h). However, inclusion of peptides, namely gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide) accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to pre-formed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 hours. Gramicidin, on the other hand, was pre-incorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g. adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.