Buffering capacity is determinant for restoring early α-synuclein aggregation.

Abstract

For disordered proteins, including α-synuclein (Syn), the aggregation of which is implicated in Parkinson's disease, it is known that at mild acidic and at the pI solution conditions the use of either strong or weak electrolytes minimized Syn aggregation. The mechanism is driven by electrostatic forces but remains, however, poorly understood. To address this issue, we used two biological buffers as weak electrolytes, at a low concentration (10mM) and monitored the aggregation of Syn solutions from pH7 to pH2, by means of light scattering techniques. When the citrate buffer was used, in which there is buffering capacity in the pH range studied, the maximum of Syn aggregation was very close to the isoelectric point (pI=4.7). When using tris-HCl, in which there is almost no buffering capacity in the pH range studied, it was for the first time observed a slow transition of the pI (of ca. 1h) from 4.7 to 4-3, for a 33.5μM protein concentration, as an example. We also observed in the protein solutions (in tris-HCl) the very early formation of large Syn aggregates. When there is buffering capacity, such as pH7, these early large Syn aggregates dissociate, followed by association/aggregation. When there is no buffering capacity, such as pH3, the referred early large Syn aggregates only dissociate. Overall, early large Syn aggregates dissociation can cause entropy in the protein solutions and Syn aggregation is only restored by the altered electrostatic forces due to the existing buffering capacity. Finally, by using an innovative strategy based in the ANS dye fluorescence intensity variation, we determined of the occurrence of the liquid-liquid phase separation process at pH7 Syn solutions.