Figure 4. AS69 inhibits $\alpha$-synuclein fibril elongation.

(a) Schematic representations of fibril elongation. (b) Change in ThT fluorescence when a 30 μM solution of monomeric $\alpha$-synuclein was incubated in the presence of 5 μM pre-formed fibrils under quiescent conditions with increasing concentrations of AS69. (c) Relative rates of fibril elongation with increasing concentrations of AS69. The solid line corresponds to a prediction based on the affinity of AS69 for monomeric $\alpha$-synuclein (240 nM, Figure 1b [Mirecka et al., 2014], see Appendix 1 for details).

Figure 4—figure supplement 1. Characterisation of $\alpha$-synuclein fibrils formed in the presence and absence of AS69 by AFM.

AFM images of 30 μM monomeric $\alpha$-synuclein incubated with 5 μM pre-formed fibrils, (a) in the absence or (b) presence of 3 μM AS69, or (c) 30 μM AS69 in 20 mM phosphate buffer at pH 6.5 under quiescent conditions at 37°C for 24 h.

Figure 4—figure supplement 2. Binding specificity determines the inhibitory activity.

The fibril elongation assay was repeated for ZA${\beta }_3$W, a binding protein for amyloid-$\beta$ peptide (Grüning et al., 2013) that is a significantly weaker $\alpha$-synuclein binder than AS69. The absence of detectable heat of binding in ITC allows only an estimate to be made for a minimal Kd of the ZA${\beta }_3$W-$\alpha$-synuclein interaction in the order of 10 μM (Mirecka et al., 2014). (a) Change in ThT fluorescence when a 30 μM solution of monomeric $\alpha$-synuclein was incubated in the presence of 5 μM pre-formed fibrils under quiescent conditions with increasing concentrations of ZA${\beta }_3$W. (b) Relative rates of fibril elongation with increasing concentrations of ZA${\beta }_3$W. For comparison to AS69, the dotted line corresponds to the solid line in Figure 4c.