Fig. 2

Fasudil delays the aggregation of α-Syn in solution through interaction with the α-Syn C-terminal domain. a ThT aggregation monitoring of a 100 μM solution of α-Syn shows a longer nucleation phase and a smaller slope in presence of a 10:1 Fasudil to protein ratio. Data are given as mean ± SD of at least three independent experiments, p < 0.001, ANOVA with Dunnett post-hoc test. Note that the ROCK inhibitor Y-27632 has no effect on the aggregation kinetics even in the same concentration. b Electron micrographs show a decreased fibril formation after 10 days of incubation in aggregation-prone conditions when Fasudil is present. Scale bars represent 200 nm. c Selected regions of 2D ¹⁵N-¹H HSQC spectra of α-Syn in the absence (black) and presence (red) of a 20:1 Fasudil to protein ratio. d Chemical shift perturbation plot of ¹⁵N/¹H resonances of α-Syn in the presence of increasing concentrations of Fasudil (Fasudil-to-α-Syn molar ratios are indicated). Note that the most affected amino acid residues are in the C-terminal region, especially Y133 and Y136. e Structural representation of α-Syn and Fasudil’s interaction through the C-terminal region (light blue), specifically in residues Y133 and Y136 (red). The N-terminal region of α-Syn is indicated in dark blue, the non-Aβ component (NAC) domain is marked purple