Fusion-peptide-induced vesicle swelling is associated with membrane surface ordering

The importance of vesicle swelling for membrane fusion has been appreciated for a long time, but the mechanism for vesicle swelling is still unknown. Our new ESR spin-labeling studies suggest a mechanism for fusion-peptide-induced vesicle swelling. It has been reported that binding of wt20 to a SOPC large unilamellar vesicle at pH 5 causes swelling and lysis of the vesicle; the vesicle swelling is weaker at pH 7 than at pH 5, but binding of mutant ΔG1 has no effect on the vesicle. We find HA2 fusion peptide wt20 increases the ordering of the headgroup region and the acyl chain region near the headgroup, but a fusion deficient mutant ΔG1 does not. That is at 25^oC and pH 5 the order parameters S₀ of a head group labeled PC, DPP-Tempo, and a chain labeled PC, 5PC, in DMPC bilayers increase significantly as the amount of wt20 vesicles exceeds the molar ratio of wt20/DMPC of 1.3 x10^-3. The increase in S₀ of 14PC, as the same amount of wt20 is added, is negligible. The ordering effect of wt20 on DMPC head-groups is stronger at pH 5 than at pH 7. However, no effect on the order of DMPC bilayers was observed after addition of ΔG1. These results are shown by figures below for DPP-Tempo. We found that addition of LysoPC into DMPC bilayers decreases the order of the head-groups of DMPC, but the effect of addition of AA is the opposite. This is correlated with reports from other studies that lysoPC inhibits but AA promotes viral membrane fusion. The results indicate that an increase in the headgroup order upon fusion peptide insertion may be important for vesicle swelling. Our results suggest the following mechanism. First, after wt20 binding to a vesicle, the increased order of the headgroups in the outer leaflet of the vesicle bilayer increases the chemical potential of water on the surface of the vesicle relative to that inside the vesicle, thus inducing water flowing from the vesicle exterior into the vesicle interior. Second, an increase in the order of the polar region in the outer leaflet of the vesicle bilayer creates an asymmetric bilayer structure which induces bending stresses in the vesicle bilayer. And finally, increased ordering of headgroups reflects stronger dipolar interactions in the polar region, which results in a reduction of electrostatic free energy that fuels the vesicle swelling.