Effect of acidic pH on the stability of α‐synuclein dimers

Abstract

Environmental factors, such as acidic pH, facilitate the assembly of α-synuclein (α-Syn) in aggregates, but the impact of pH on the very first step of α-Syn aggregation remains elusive. Recently, we developed a single-molecule approach enabling us to measure directly the stability of α-Syn dimers. Unlabeled α-Syn monomers were immobilized on a substrate, and fluorophore-labeled monomers were added to the solution to allow them to form dimers with immobilized α-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 α-Syn dimers. The experiments were performed at pH 5 and pH 7 for the wild-type α–Syn and two mutants containing familial type mutations E46K and A53T. We demonstrate that decrease of pH resulted in more than 3-fold increase in the α-Syn dimers lifetimes with some variability between the α-Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96–140 of α-Syn and this electrostatic effect facilitates the association of the two monomers. Given the fact that dimerization is the first step of α-Syn aggregation, we posit that the electrostatic effect contributes to accelerating α-Syn aggregation at acidic pH.

Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: what you see is not always what you get

“…It was suggested that these observations could be explained by the possibility that the dimerization propensity of the asyn proteins to form dimers does not reflect their oligomerization and aggregation properties (Eckermann et al, 2015). However, this interpretation is not consistent with extensive biophysical studies using alternative approaches that show significant differences in the propensity of a-syn with the various PD-linked a-syn mutants to form oligomers (Winner et al, 2011) and dimers (Janowskaet al, 2015;Lv et al, 2016). In addition, several studies have shown significant differences in the lifetime and distribution of different types of dimers formed by wild-type a-syn and PD-linked mutants (Krasnoslobodtsev et al, 2013;Krishnan et al, 2003;Lv et al, 2015).…”

Spontaneous self-assembly of amyloid β (1–40) into dimers

“…Recent studies have demonstrated that single-molecule approaches are powerful methods to study oligomers. [43][44][45][46] Single-molecule techniques, such as AFM, 11,[47][48][49][50][51] tethered approach for probing inter-molecular interactions (TAPIN), 52,53 and FRET, 33 have shown that the early-stage oligomers exhibit prolonged lifetimes and stabilities. Novel features of the interaction and self-assembly of Ab40 and Ab42 peptides were determined using single-molecule AFM-based force spectroscopy.…”

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

“…39 More recently, the design of peptides that specifically bind to strands F and H of TTR have been shown to effectively work as amyloid inhibitors by capping those strands and preventing head-to-head association of TTR monomers. 17 The particular relevance of dimers over other similar low molecular weight aggregates (eg, trimers, tetramers, and pentamers) is not exclusive to TTR and has been reported in a variety of amyloidogenic systems such as peptides Aβ 40 and Sup35, 41 as well as proteins, including αsynuclein 42,43 and β2-microglobulin. 44 Thus, it is conceivable that the arrangement of monomers along the aoTTR annuli might result in alternating strongly and weekly interacting monomer-monomer interfaces, with the strong interfaces associated with dimer formation and weak interfaces defining the dimer outer limits.…”