Abstract
A methyl-beta-cyclodextrin-induced lipid exchange technique was devised to prepare small unilamellar vesicles with stable asymmetric lipid compositions. Asymmetric vesicles that mimic biological membranes were prepared with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-beta-cyclodextrin exchange step. In asymmetric vesicles composed of SM outside, DOPC inside (SMo/DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in symmetric vesicles with the same overall lipid composition. Analogous behavior was observed in vesicles containing cholesterol. This shows that an asymmetric lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asymmetric vesicles containing approximately 25 mol % cholesterol formed ordered domains more thermally stable than those in asymmetric vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Additional studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asymmetric vesicles represents an important improvement in model membrane studies and should find many applications in the future.
Highlights
Perhaps been the most useful model membrane system
Asymmetric vesicles that mimic biological membranes were prepared with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet
In asymmetric vesicles composed of SM outside, DOPC inside (SMo/ DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/ 2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in symmetric vesicles with the same overall lipid composition
Summary
The ability to exchange lipids in one leaflet of the bilayer can provide a method to prepare asymmetric vesicles with controlled asymmetry [38, 39]. This procedure is based on the observation that methyl--cyclodextrin (MCD) binds phospholipids at very high MCD concentrations [42, 43] Using this method asymmetric vesicles were prepared with an external leaflet rich in sphingomyelin (SM) and an internal leaflet rich in PE and PS, similar to eukaryotic plasma membranes. The physical properties of these vesicles reveal some important differences and similarities between symmetric and asymmetric bilayers Overall it appears that the type of asymmetry found in eukaryotic cell membranes is not a barrier to raft formation and, even more importantly, that the stabilizing effects of cholesterol upon raft formation are not restricted to symmetric membranes. Asymmetric vesicles prepared by this method should aid many studies of the role of lipid asymmetry in membrane structure and function
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