Kinetics of lipid rearrangements during poly(ethylene glycol)-mediated fusion of highly curved unilamellar vesicles

uIn an effort to increase our understanding of the molecular rearrangements that occur during lipid bilayer fusion, we have used different fluorescent probes to characterize the lipid rearrangements associated with poly(ethylene glycol) (PEG)-mediated fusion of DOPC:DL18;3PC (85:15) small, unilamellar vesicles (SUVs). Unlike in our previous studies of fusion kinetics [Lee, J., and Lentz, B. R., Biochemistry 36, 6251-6259], these vesicles have mean diameters of 20 nm compared to 45 am. Surprisingly, we found significant inter-vesicle lipid mixing at 5 wt % PEG, well below the PEG concentration required (17.5 wt %) for vesicles fusion. Lipid movement rate between bilayers (or inter-leaflet movement) increased abruptly at 10 wt % PEG, and the rate of lipid mixing increased thereafter with increasing amounts of PEG. The characteristic time of lipid mixing between outer leaflets (tau approximate to 24 s) was comparable to that observed at and above PEG concentrations needed to induce fusion (17.5 wt %) of either 20 or 45 nm vesicles. We also found that slower lipid mixing (tau approximate to 267 s) between fusing vesicles occurred on the same time scale or slightly faster than vesicle contents mixing (tau approximate to 351 s). In addition, our measurements showed that lipids redistributed across the bilayer on a time scale just slightly faster than pore formation (tau approximate to 217 s). This is the first demonstration of trans-bilayer movement of lipids during fusion. We also found that water was excluded from the bilayer (tau approximate to 475 s) during product maturation. These observations suggest that fusion in smaller vesicles (similar to20 nm) proceeds via a multistep mechanism similar to that we reported for somewhat larger vesicles, except that two intermediates are no longer clearly resolved.