Figure 2. Thermodynamic profiling uncovers key features stabilizing S320F295–330 amyloid fibril.
Calculated ΔΔG value of each amino acid in chain A (a) and chain B (b) calculated from in silico alanine mutagenesis for assemblies with increasing numbers of layers n=3, 6, 9 and 12. The ΔΔG values are colored from black (n=3) to grey (n=12). (c) Mapping per residue ΔΔG values onto chains A and B of the S320F295–330 cryo-EM structure uncovers hotspot residues in chains A and B at the N-terminus and C-terminus (>1 SD above the mean). Structures are shown as a single layer with the backbone shown as ribbons and the C-β of each residue is shown as a sphere. Each amino acid is colored by calculated ΔΔG from n=9 layer analysis. (d) The per residue ΔΔG values are shown mapped on a model of the S320F295–330 amyloid to highlight residues that stabilize the amyloid fold: (1) VQIVYK-VQIVYK, (2) C322-C322 disulfide and (3) I328-I328. Backbone is shown as ribbon and side chains are shown as sticks. (e-f) ThT fluorescence aggregation assay of S320F295–330 (−), S320F295–330 (TCEP), S320F295–330 C322S carried out at 10 μM (white), 15 μM (light grey) and 20 μM (grey). Data is shown as t1/2max (e) and ThT fluorescence amplitude (f) parameters derived from a nonlinear regression fit to each aggregation curve. Data is presented as an average of three replicates with error bars as standard error.