Figure 4

Effect of Munc18-1 on the conformation of syntaxin-3b and its mutants. (A) The structure of syntaxin/Munc18 complex showing the closed conformation of syntaxin (PDB ID: 3C98). Protein domain diagram of syntaxin-3b constructs used in this experiment (bottom panel). (B) Schematic of a surface-tethered syntaxin-3b molecule labeled with FRET dye pairs on a functionalized surface of the microscope slide in a closed conformation and locked in a closed conformation when bound to 1 μM Munc18-1 in solution. (C) Representative single-molecule fluorescence intensity time traces of WT (left panels), T14E (middle panels) and LE (right panels) mutation on syntaxin-3b in the presence of 1 μM Munc18-1 in solution. The donor and acceptor intensities were converted to FRET efficiency time traces (bottom panels). (D) smFRET efficiency histograms of wild-type, T14E and LE mutations on syntaxin-3b in the presence of 1 μM Munc18-1 in solution. (E) Percent closed populations of syntaxin-3b WT, T14E, and LE mutant in the presence of 1 μM Munc18-1 in solution were extracted from (D) by fitting two Gaussian functions to the FRET efficiency histograms. Shown are means ± SD (n = 3). (F–G) A pulldown assay was conducted using biotinylated syntaxins in complex with Munc18-1 as bait on neutravidin coated beads to estimate the efficiency of binary t-SNARE complex formation with SNAP-25. Proteins bound to the beads were confirmed by SDS-PAGE. SNAP-25 bands, indicating the binary t-SNARE complex formation, as well as Munc18-1 bands were analyzed using ImageJ software (NIH, Bethesda, MD). Shown are means ± SD (n = 3). Original SDS-PAGE gels are presented in Supplemental Fig. 5.