Figure 1.
Correlation between aggregation propensity and disorder scores for S100 proteins.
Plot of weighted average aggregation propensity (average Zagg) versus average disorder (PONDR) scores computed using the Zyggregator and VSL2P algorithms, respectively. Squares stand for to the studied S100 proteins and diamonds for well-known proteins with bona fide disordered regions (Aβ, PrP, amylin and ABri, as compiled in [18]. The threshold for high aggregation propensity is 1 (Zagg>1) and for intrinsic disorder is 0.5 (PONDR>0.5).
Figure 2.
Position specific analysis and structural mapping of tandem disordered and aggregation-prone segments in S100 proteins.
(A) The top scheme is a linear representation of the secondary structure elements in S100 proteins. The red bars in the plot stand for average aggregation propensity within S100 proteins. The blue curve was redrawn from [16] and represents the average disorder PONDR VSL2P score for S100 proteins. The threshold for high aggregation propensity is 1 (Zagg>1) and for intrinsic disorder is 0.5 (P>0.5). Below is a multiple sequence alignment of the studied S100 proteins. The residues with values above the thresholds for aggregation and intrinsic disorder scores for individual proteins are highlighted in red and blue, respectively. The residues with values above both thresholds are highlighted in gray. The Ca2+-coordinating residues in the S100 and canonical EF-hands are highlighted in bold. (B) Representation of high aggregation propensity regions (red) of S100 proteins located in helices α1 and α4 (Zagg>1), which are contiguous to locally disorder-prone segments (blue) mainly in helices α2 and α3 and on the hinge region (PONDR>0.5). (C) Representation of a S100 dimer highlighting the segments with high propensity within helices α1 and α4 located at the dimer interface, to illustrate the structural protection afforded by dimer assembly.
Figure 3.
Fluorescence intensity of ThT and ANS bound to S100 proteins.
The bars denote ThT (dark grey) and ANS (light grey) fluorescence upon diluting the different S100 proteins (1 mg/ml) in acidic buffer (glycine pH 2.5) at up to 30 min.
Figure 4.
ThT binding kinetics and dot blot analysis of amyloid epitopes.
A. S100 proteins (1 mg/mL) were incubated at pH 2.5 and 37°C during 160 h without agitation in the presence of ThT. The enhancement of ThT fluorescence at 480 nm was used to monitor amyloid formation, and the different proteins are color coded in the figure: S100A2 (dark blue), S100A3 (red), S100A4 (green), S100A6 (purple), S100A8/A9 (orange), S100A12 (light blue), S100B (pink). S100A12 is the protein undergoing faster ThT binding (kapp= 0.38 h-1), S100A6 (kapp= 0.14 h-1) and S100A4 (kapp= 0.09 h-1) are slower, with comparable fibrillation rates, and finally S100A8/A9 has the slowest fibrillation rate (kapp= 0.03 h-1). B. After incubation under amyloidogenic conditions, the conformation of S100 proteins was probed in a dot blot assay with the conformation-sensitive antibodies OC (anti fibrillar oligomers and fibrils) and A11 (anti pre-fibrillar oligomers). α-synuclein aggregates (oligomers and fibrils) and Aβ (fibrils) are included as controls.
Figure 5.
TEM imaging of S100 β-aggregates and amyloids.
TEM images of S100 proteins (1 mg/mL) upon 160 h incubation at pH 2.5 and 37°C. (A) S100A2, (B) S100A3, (C) S100A4, (D) S100A6, (E) S100A8/A9, (F) S100A8/A9, magnification of panel E, (G) S100A12, (H) S100A12, magnification of panel G, (I) S100B and (J) S100B, magnification of panel I. Scale bars: 200 nm (panels A, B, C and D); 100 nm (panels E, G and I) and 50 nm (panels F, H and J). See text for details.