Acyl Chain Unsaturation and Vesicle Curvature Alter Outer Leaflet Packing and Promote Poly(ethylene glycol)-Mediated Membrane Fusion

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Abstract
The poly(ethylene glycol) (PEG)-induced fusion of sonicated, unilamellar vesicles (SUV) and large, unilamellar vesicles (LUV) composed of a variety of phosphatidylcholine species was compared using two assays for the mixing and leakage of internal vesicle contents. In the first [Lentz et al. (1992) Biochemistry, 31, 2643], disodium 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) fluorescence is quenched by co-encapsulated N,N'-p-xylylenebis(pyridinium bromide) (DPX). For this assay, interference by the fluorescence of impurities in PEG demands that the PEG content of the sample be reduced by dilution before measurements are taken. The second assay [Viguera et al. (1993) Biochemistry, 32, 3708] monitors the fluorescence of Tb3+ complexed with dipicolinic acid (DPA) directly in concentrated PEG solutions. The two assays gave identical fusion profiles for egg PC SUVs treated with increasing concentrations of PEG, demonstrating that fusion occurs in the dehydrated state in the presence of PEG and does not require dilution. Comparison of results obtained with lipid species of varying degrees of unsaturation incorporated into either SUV or LUV suggested that acyl chain unsaturation and high membrane curvature combine to favor fusion of pure phosphatidylcholine membranes. There was a clear correlation between the fluorescence lifetime or the order parameter of the membrane probe 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in different membranes and the concentration of PEG needed to induce fusion of these membranes. However, the ratios of TMA-DPH lifetimes measured in D2O versus H2O buffers were the same for different lipid species, indicating that probe penetration was not very different for different lipid species. The results suggest that the combined effect of high membrane curvature and extensive chain unsaturation is an enhanced rate of lipid motion in the upper region of the bilayer, reflective of decreased packing density in the outer leaflet of unsaturated SUV bilayers, probably allowing for enhanced water penetration leading to an enhanced probability of fusion.