Lipid–Substrate Interactions Lead to Bilayer Asymmetry

Lipid asymmetry was probed in supported lipid bilayers (SLBs) formed by vesicle fusion on planar glass substrates and protein-coated substrates. Leaflet distribution was determined via fluorescence microscopy following bilayer unzipping. Each SLB was primarily composed of phosphatidylcholine (PC) with 0.5 mol % of a test lipid containing a tail-labeled probe. At equilibrium, the results revealed that 75% of labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) lipids partitioned into the lower leaflet adjacent to the glass surface. This preferential partitioning further increased when experiments were conducted in D₂O rather than an H₂O buffer, a finding consistent with favorable hydrogen bond (H-bond) formation between surface silanols on the glass support and the headgroups of PS and PE lipids. In fact, H-bonding was the dominant factor in determining the interleaflet distribution. Varying the salt concentration could modulate the PS fraction in only the lower leaflet by a small amount, ±3%. A tail-labeled PC probe, which was unable to donate an H-bond to the surface, showed no preference for the lower leaflet once its equilibrium distribution was achieved. Finally, the 2D diffusion of the PS and PE probes, but not PC, was strongly dependent on the number of available H-bonding sites on the substrate surface. These results are reminiscent of the lipid asymmetry observed in the plasma membranes of living cells, where PS and PE are similarly partitioned into the inner leaflet, suggesting that the H-bonding environment in the cytoplasmic versus extracellular region may play a significant role in governing lipid asymmetry in vivo.