Munc18-1 and complexin-1 can bind simultaneously to the SNARE complex. (A) Sample traces of the methyl regions of 1D ¹³C-edited ¹H-NMR spectra of 2 µM Munc18-1 in the absence or presence of 2 µM of unlabeled SNARE complex or 2 µM of unlabeled SNARE complex plus 2 µM ¹⁵N-labeled complexin-1. (B) Sample traces of 1D ¹⁵N-edited ¹H-NMR spectra of 2 µM ¹⁵N-labeled complexin-1 in the absence or presence of 2 µM of unlabeled SNARE complex or 2 µM of unlabeled SNARE complex plus 2 µM ¹³C-labeled Munc18-1. (A and B) The spectra on the right were acquired with the same sample. ppm, parts per million. (C) Gel filtration on a Superdex S200 (10/300GL) column of Munc18-1, complexin-1, SNARE complex (SC), and mixtures of the SNARE complex with Munc18-1, complexin (Cpx), or both. The SNARE complexes used for all of these experiments contained syntaxin-1(2–253), synaptobrevin-2(29–93), SNAP-25(11–82), and SNAP-25(141–203).