Fig. 3: Biophysical and structural characterisation of sc-CC-7 de novo proteins.
From: Rationally seeded computational protein design of ɑ-helical barrels
a, Helical-wheel representation for part of a parallel single-chain α-helical barrel showing KIH packing for the buttressing helices (shaded red) and the inner barrel (shaded blue): red, a sites; green, d sites; magenta, g sites; and cyan, e sites; N and C labels refer to the termini of the helices closest to the viewer. b, Sequence pileups and registers for the inner (blue register) and buttressing (red register) helices of sc-CC-7-LI. c,d, Circular dichroism spectrum recorded at 5 °C (c) and thermal-response curve (d) for sc-CC-7-LI. e, AUC sedimentation velocity data for sc-CC-7-LI fitted to a single-species model, which returned MW = 37.4 kDa (monomer). f, Fitted binding data of DPH to sc-CC-7-LI, which returned Kd = 3.8 ± 0.8 µM. Fitted data are the mean and s.d. of three independent repeats. g, SEC-SAXS data fitted using the final AlphaFold2 model and FoXS (χ2 = 1.43)57,58. h, X-ray crystal structure of sc-CC-7-LI at a 2.5-Å resolution (PDB ID, 8QAI). Coiled-coil regions identified by Socket2 (ref. 72) (packing cutoff, 7 Å) are colored as chainbows from N termini to C termini (blue to red). i, A slice through the structure of a heptad repeat showing KIH packing with a-type (red) and d-type (green) knobs. j, Overlay of the middle helical turns from the sc-CC-7-LI structure (cyan) and the final AlphaFold2 model (magenta) (RMSDbb = 0.433 Å). The conditions were as follows: circular dichroism spectroscopy, 5 µM protein in PBS, pH 7.4; AUC, 25 µM protein in PBS, pH 7.4; DPH binding, 0–24 µM protein in PBS, pH 7.4, final concentration was 0.5 µM DPH (5% v/v DMSO); SEC-SAXS, 10 mg ml−1 protein in PBS, pH 7.4.
