Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays. •SNARE-driven membrane fusion was analyzed by dynamic light scattering (DLS).•Two vesicles with the same hydrodynamic diameter D resulted in 2D after full fusion.•Hemifusion and docking resulted in 2D and 2D + intermembrane gap, respectively.•DLS analysis indicated that delphinidin and cyanidin prevented vesicle docking and myricetin stopped membrane fusion at hemifusion state.•Effect of flavonoids on fusion by SNARE was consistent between analysis techniques.