Fig. 4. Effects of environmental stimuli on α-synuclein microscopic steps of aggregation. (a) Legend of the condition of incubation used in the present work. (b) Slope of the ThT vs., CD signal at 45 μM. (c) Volume of the fibrillar aggregates formed under incubation in each condition after 30 days (solid points) and 54 days (transparent points). (d) A double logarithmic plot of the half time of aggregation versus initial protein concentration. (e) Rates of primary nucleation (blue) and secondary (red) processes at 30 μM monomer concentration obtained from global fitting of the aggregation kinetics at the different conditions to an integrated rate law. (f) Schematic representation of the effect of hydrodynamic mixing and air–water interface on primary nucleation and secondary process during aggregation of α-synuclein. Primary nucleation is significantly increased in the presence of a moving air–water interface, whereas secondary processes are increased by rotating in the presence of an air–water interface or shaking.