Fig. 2: Characterization of SP9.
From: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides
a, Magnified view of the primary interface between the neuronal SNARE complex (VAMP-2 (blue), Stx1 (red) and SNAP-25A (green)) and the C2B domain of Syt1 (orange) (Protein Data Bank (PDB): 5W5C), indicating the region (yellow) that corresponds to the stapled peptide SP9 with staples shown as dumbbells. b, Schematic of the synthesis of SP9. Hydrocarbon-stapled peptides are formed by cross-linking residues at the specified positions. c, Sequences of peptides. S5 indicates S stereochemistry at the α-carbon, with 5 carbon atoms in the olefinic side chains. The superscripts denote the start and end positions of the SNAP-25A sequence. d, Circular dichroism (CD) spectra of 100 mM peptides measured at pH 7.4 and at 25 ± 1 °C. e, The percentage of α-helical content in these peptides was estimated by dividing the mean residue ellipticity [φ]222obs by the reported [φ]222obs for a model helical decapeptide. f, Interactions between Cy3-labelled SP9 or P0 and unlabelled Syt1 C2B, the quintuple Syt1 C2B(QM) mutant and Syt2 C2B as measured by bulk fluorescence anisotropy (Methods). Data are mean ± s.e.m. along with individual data points from n = 3–7 independent experiments. Hill equations were fit to estimate the dissociation constant Kd, where the Hill coefficients were constrained to 1. g, Peptide conformations (colours) after five independent 1 μs molecular dynamics simulations of SP9–Syt1 C2B (left) and P9–Syt1 C2B (right) superimposed onto the structure of the primary interface (grey). The simulations started from a conformation (Extended Data Fig. 2f, g) that was derived from the crystal structure PDB 5W5C (Supplementary Videos 1 and 2). For one simulation of P9–Syt1 C2B, the P9 peptide dissociated around 168 ns.
