Fig. 3: Conformational transition of VAMP2 on membrane subdomains of synaptic vesicles.

a Scheme of experimental design for the structural study of VAMP2 on SV membrane. Upper: SVs isolated from mouse brain were used for NMR titration of VAMP2(1–96) at the physiological ratio. Middle: natural SV membranes were divided into lipid-raft and non-raft membranes by sucrose gradient sedimentation which were analyzed by quantitative lipidomic profiling. Lower: lipid-raft- and non-raft-mimicking vesicles were reconstituted with natural-sourced lipids to titrate VAMP2(1–96). b Residue-resolved NMR signal intensity ratios (I/I₀) of VAMP2(1–96) titrated by SVs to that in solution. The molar ratio of SV to VAMP2(1–96) is indicated. c Left: distribution of endogenous VAMP2 on SV membrane. SV membranes were fractionated into 13 layers which were collected as lipid raft (layer 3) and non-raft (layers 9–12) according to flotillin2 (lipid raft membrane protein) and rabphilin3A (non-raft membrane protein). Right: lipid compositions of lipid-raft and non-raft membranes by MS-based lipidomic profiling. Chol: cholesterol; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PS: phosphatidylserine; PI: phosphatidylinositol; PG: phosphatidylglycerol; PA: phosphatidic acid; Cer: ceramide; SM: sphingomyelin; TG: triacylglycerol. d Residue-resolved NMR signal intensity ratios (I/I₀) of VAMP2(1–96) titrated by lipid-raft-mimicking (blue) or non-raft-mimicking (red) vesicles to that in solution at indicated lipid/protein molar ratios. e Scheme of single-vesicle docking assay. A saturated layer of DiD-labeled (red) t-SNARE vesicles carrying syntaxin-1a and SNAP25 was immobilized on the imaging surface. Free DiI-labeled (green) v-SNARE vesicles, reconstituted with full-length VAMP2, were injected into the system. Green laser illumination imaged the v-vesicles that docked on t-vesicles through SNARE complex formation. f Images on the right are representative fluorescence images of the single-vesicle docking assay. The bar graph on the left shows the numbers of lipid-raft- and non-raft-mimicking v-vesicles that docked on t-vesicles. Error bars are standard deviations from 20 random imaging locations in the same sample channel. *** indicates p-value < 0.001 by One-way analysis of variance (ANOVA) with Tukey Test. Source data are provided as a Source Data file.