Purified HMunc18c is monomeric in solution.

<p><b>A</b>. Elution profile of purified HMunc18c on a calibrated analytical size exclusion chromatography column (S200 10/300 GL). HMunc18c eluted at a volume consistent with a ∼70 kDa protein. Peak fractions were analysed on 4–12% gradient SDS-PAGE (inset). <b>B</b>. Elution profile of HMunc18c examined by SEC-MALS. The horizontal blue line corresponds to the SEC-MALS calculated mass (right axis) plotted with the refractive index indicating the peak (left axis) of the protein in the sample (68,200 Da ±0.5%).</p

Isothermal titration calorimetry data.

<p>The raw data (upper part of each panel) and integrated normalized data (lower part of each panel) are shown from ITC experiments between HMunc18c or Munc18a-His and cognate/non-cognate Sx partners.</p

Munc18 proteins bind non-cognate Sxs.

<p>Sx1_1-261-His or Sx4_1-275-His were incubated with detagged Munc18a or Munc18c for 2 h, 24 h or 48 h before the Sx was immobilized onto TALON^™ Co²⁺ affinity beads for 1h and then washed. Samples of the beads were then run on SDS-PAGE and stained with Coomassie Blue to determine if detagged Munc18 had been pulled down by cognate and non-cognate Sx partners. Detagged Munc18a and Munc18c were also incubated for the same time periods on beads without bound Sx to monitor non-specific binding (control lanes). Solid vertical lines on the gel image denote the removal of intervening lanes or placing two different gels adjacent to each other.</p

Reported Munc18c expression and purification.

<p><b>NR*</b>- Not reported, <b>FL</b>- full-length, <b>PDA</b>-pull down assays; <b>IP-</b> immunoprecipitation; <b>ITC</b>- isothermal titration calorimetry; <b>SAXS</b>-small angle X-ray scattering.</p

Expression optimization of codon-optimized full-length HMunc18c in <i>E. coli</i> cultures.

<p>Different media, cell expression strains and expression temperatures (as indicated) were used to optimise yield of HMunc18c using 1 L cultures. All cultures were grown at 37°C until OD₆₀₀ reached 0.5–0.6 and then either induced with 1 mM IPTG (LB and TB) and/or temperature lowered. <b>A.</b> BL21 strain, LB Media, 25°C. <b>B.</b> BL21 strain, LB media, 20°C. <b>C.</b> BL21 strain, TB media, 25°C. <b>D.</b> BL21 strain, TB media, 20°C. <b>E.</b> BL21 strain, ZYP-5052, 25°C. <b>F.</b> BL21 strain, ZYP-5052, 20°C. <b>G.</b> BL21 strain, ZYP-5052, 16°C. The black arrow indicates the expected band for HMunc18c.</p

Purification of full-length Munc18c from test expressions in <i>E. coli</i> cultures.

<p>Coomassie stained SDS-PAGE showing purification of Munc18c from test expressions. <b>A.</b> HMunc18c<i>_w</i>, using conditions similar to those described in Brandie et al., (2008). Wash and elution fractions from beads are shown. <b>B.</b> HMunc18c, using the same conditions.</p

Schematic representation of the protein constructs used in this study.

<p><i>In vivo</i> full-length Sx consists of an N-peptide preceding an N-terminal α-helical bundle (the H_abc domain), a SNARE motif (the H3 helix) and a C-terminal transmembrane region. We used soluble Sx1 and Sx4 constructs lacking the transmembrane domain for experiments reported here. ΔN indicates Sx constructs lacking the N-peptide. Munc18 and SNAP25 and VAMP2 constructs used in these experiments are also shown. The positions of engineered fusion tags and protease cleavage sites are as indicated.</p

Thermodynamic parameters of Munc18c/Sx4 interactions (values are shown as a mean from four sets of different experiment ± SEM).

<p>Thermodynamic parameters of Munc18c/Sx4 interactions (values are shown as a mean from four sets of different experiment ± SEM).</p

Purification of recombinant HMunc18c expressed in <i>E. coli</i> cultures.

<p><b>A.</b> SDS-PAGE analysis of HMunc18c purification steps. Elution fraction from IMAC, labelled, was injected onto SEC and eluted as shown in <b>Lanes 1–6</b> (which correspond to the labelled fractions in panel B). <b>B.</b> Elution profile of HMunc18c from SEC. Peak fractions were pooled and injected onto a MonoS column. <b>C.</b> SDS-PAGE of fractions from the MonoS purification step, showing separation of the protein from lower molecular weight contaminants. <b>D.</b> Elution profile of HMunc18c from MonoS.</p

Non-cognate Munc18 interactions with SNARE complexes.

<p>(A) Cognate and non-cognate Munc18 proteins were first incubated with Sx1_1-261-His to form the Munc18:Sx1_1-261-His binary complex in the presence of beads. The beads were washed, then incubated with the Sx1 SNARE partners, SNAP25 and VAMP2, overnight at 4°C before SNARE complex formation was assessed by the presence of SNAP25 and VAMP2 on the beads (analysed by SDS-PAGE with Coomassie Blue staining). (B) Pre-formed SNARE complex comprising Sx1_1-261-His:SNAP25:VAMP2 was captured by beads and then incubated with detagged Munc18a or detagged Munc18c for 2 h at 4°C. The SNARE complex was captured using affinity beads, and the presence of bound Munc18 was evaluated by SDS-PAGE analysis and Coomassie Blue staining. Each image shown is representative of multiple replicate experiments, and the solid lines through the gel indicate where images of different gels have been joined.</p