Effect of Acidic pH on the Stability of α-Synuclein Dimers

Environmental factors, such as acidic pH, facilitate the assembly of a-synuclein (a-Syn) in aggregates, but the impact of pH on the very first step of a-Syn aggregation remains elusive. Recently, we developed a single-molecule approach that enabled us to measure directly the stability of a-Syn dimers. Unlabeled a-Syn monomers were immobilized on a substrate, and fluorophore-labeled monomers were added to the solution to allow them to form dimers with immobilized a-Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single-molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of a-Syn dimers. The experiments were performed at pH 5 and 7 for wild-type a-Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the a-Syn dimers lifetimes with some variability between the a-Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96-140 of a-Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of a-Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating a-Syn aggregation at acidic pH. (C) 2016 Wiley Periodicals, Inc.