Cross-linking of AG22 constructs with 14-3-3.

<p><b>A.</b> Cross-linking of truncation constructs AG22 (1–422) S16D/S411D, AG22 (38–422) (S411D) and AG22 (1–145) S16D with 14-3-3. For each cross-linking reaction, the mixture of AG22 protein construct and 14-3-3 or the individual AG22 proteins was incubated with BS3 for 10 min at room temperature. The mixtures before and after cross-linking were analyzed by SDS-PAGE and visualized by Coomassie Blue staining. Lanes on the left show BS3 cross-linking results with 14-3-3; lanes on the right show BS3 cross-linking results for AG22 proteins without 14-3-3. *indicates the cross-linked complex of 14-3-3 with AG22 (1–422) S16D/S411D; ^§the cross-linked complex of 14-3-3 with AG (38–422) S411D and ^#the cross-linked complex of 14-3-3 with AG22 (1–145) S16D. <b>B.</b> No detectable cross-linking was observed for AG22 (167–422) S411D and 14-3-3. The arrow indicates the position of the band (90 kDa) expected if AG22 (164–422) S411D (MW, 31.3 kDa) and 14-3-3 (monomer MW, 29.3 kDa) had formed a cross-linked complex. Cross-linking experiments shown in panels A and B are representative of three replicates.</p

MS identification of peptide sequences cross-linked in the AG22 (1–422) S16D/S411D:14-3-3 complex.

<p>MS identification of peptide sequences cross-linked in the AG22 (1–422) S16D/S411D:14-3-3 complex.</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

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

SEC analysis of AG22 (1–422) S16D/S411D and 14-3-3.

<p><b>A.</b> SEC elution profiles of the cross-linked (black) and uncross-linked (gray) complexes. The cross-linked complex of AG22 (1–422) S16D/S411D and 14-3-3 eluted at 12 mL from a Superdex S200 (10/300 GL) column, while the uncross-linked complex eluted at 13.5 mL. Absorbance (mAU) at 280 nm was monitored. Elution volumes of molecular mass standards are indicated at the top of the panel. <b>B.</b> Elution fractions from gel filtration were analyzed by SDS-PAGE and stained with Coomassie Blue.</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

ITC derived thermodynamic parameters for Munc18:Sx-His and Munc18:ΔNSx-His interactions.

<p>Values reported are average and standard deviation from at least three experiments.</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

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