Phase Separation and Aggregation of α-Synuclein Diverge at Different Salt Conditions.

The coacervation and structural rearrangement of the protein alpha-synuclein (αSyn) into cytotoxic oligomers and amyloid fibrils are considered pathological hallmarks of Parkinson's disease. While aggregation is recognized as the key element of amyloid diseases, liquid-liquid phase separation (LLPS) and its interplay with aggregation have gained increasing interest. Previous work showed that factors promoting or inhibiting amyloid formation have similar effects on phase separation. Here, we provide a detailed scanning of a wide range of parameters including protein, salt and crowding concentrations at multiple pH values, revealing different salt dependencies of aggregation and phase separation. The influence of salt on aggregation under crowded conditions follows a non-monotonic pattern, showing increased effects at medium salt concentrations. This behavior can be elucidated through a combination of electrostatic screening and salting-out effects on the intramolecular interactions between the N-terminal and C-terminal regions of αSyn. By contrast, we find a monotonic salt dependence of phase separation due to the intermolecular interaction. Furthermore, we observe the time evolution of the two distinct assembly states, with macroscopic fibrillar-like bundles initially forming at medium salt concentration but subsequently converting into droplets after prolonged incubation. The droplet state is therefore capable of inhibiting aggregation or even dissolving the aggregates through a variety of heterotypic interactions, thus preventing αSyn from its dynamically arrested state.