Constructs for bacterial expression of full-length rat Munc18-1, squid Munc18-1, or fragments of rat synaptobrevin-2 (residues 29–93), human SNAP25 (residues 11–82 and 141–203), the cytoplasmic domain (residues 2–253) of rat syntaxin-1A, its N-terminal region (residues 1–180) or its SNARE motif (residues 191–253), as well as the syntaxin-1A(2–253) L165E,E166A mutant (LE mutant), were described previously10 (link), 13 (link), 44 (link), 52 (link). The synaptobrevin(29–93) (S61C), syntaxin-1A(191–253) (R210E), syntaxin-1A(2–253) (R210E), syntaxin-1A(2–253) (C145S, S249C), syntaxin-1A(2–253) (L165E,E166A, C145S, S249C) and syntaxin-1A(2–253) (R210E, C145S, S249C) mutants were generated from the corresponding parent fragments using QuickChange site-directed mutagenesis kit (Stratagene). The vector to express rat Munc13-1(859–1407,1453–1531) fragment (MUN*) was prepared from the previously described Munc13-1(859–1531) fragment32 (link) using standard molecular biology techniques. All proteins were expressed as GST fusions, isolated by affinity chromatography, and purified by gel filtration and/or ion exchange chromatography as described10 (link), 13 (link), 32 (link), 52 (link). Isotopic labeling was performed using well-established procedures9 (link), 58 (link). Protein concentrations were determined by UV absorbance. Most experiments involving Munc18-1 were performed with rat Munc18-1. However, because of the limited solubility of rat Munc18-1, binding experiments where isolated Munc18-1 had to be added at concentrations above 20 μM were performed with squid Munc18-1, which is more soluble and has a 66.4% sequence identity with rat Munc18-1, having very similar biochemical properties44 (link).