Fig. 4: The impact of trimer and lateral interface variants on solution-phase SNARE assembly.

A Solution FRET experiments measuring N-terminal SNARE assembly between SNAP25 (Q20C-AF647, red) and the cytoplasmic domain of VAMP2 (CDV) (S28C-AF568, green) were measured on a plate reader excited at 565 nm and emission recorded at 635 nm. Fluorescence was measured after the addition of Munc18-1/Syntaxin 1 (gray) in the presence and absence of Munc13C (blue). B Average FRET traces recorded over 90 min after the addition of Munc18-1/Syntaxin 1 protein in the presence (black) and absence (blue) of wild-type Munc13C. Traces are mean ± SEM for N = 6 replicates. No increase in FRET was observed in the absence of Munc18-1/Syntaxin 1 (red). C, D Inclusion of unlabeled CDV inhibited SNARE assembly by 76.4 ± 1.9% (green) with **p < 0.01 using the Student’s t-test (one-tailed). Traces are mean ± SEM (N = 6). SNAREs only (black) are the same data shown in (B) for—Munc13. E Average initial SNARE assembly rates for wild-type Munc13C and all trimer and lateral interface mutant variants normalized by SNARE assembly in the absence of Munc13C. Data were presented as a mean ± SEM from N = 6 experiments. Significance was tested using Tukey–Kramer to account for multiple comparisons. ** indicates a significant difference (p < 0.01) compared to the absence of Munc13C but not wild-type Munc13C. ## indicates a significant difference (p < 0.01) with wild-type Munc13C but not the absence of Munc13C (exact p values are indicated over the bars).