Formation kinetics and physicochemical properties of mesoscopic Alpha-Synuclein assemblies modulated by sodium chloride and a distinct pulsed electric field.

Alpha-Synuclein (ASN), a presynaptic protein, has been widely reported to form amyloid-rich hydrogel clusters through liquid-liquid phase separation (LLPS) and liquid-to-solid transition. However, in-depth investigations about the parameters that influence the assembling kinetics, structures, and physicochemical properties of intermediate ASN assemblies are still missing. Therefore, we monitored for the first time the assembling and ordering kinetics of ASN by polarized/depolarized light scattering (DLS/DDLS) under the effect of ionic strength and a pulsed electric field (EF), followed by characterizing the resultant ASN assemblies applying thermostability assays, fluorescence/autofluorescence assays, and TEM. The underlying molecular mechanism was discussed based on experimental evidence. Results showed that in the presence of 150-250 mM NaCl, monomeric ASN is highly soluble in a temperature range of 20-70 °C and could form dissoluble liquid dense clusters via LLPS in crowded environments, while the ionic strength of 50 mM NaCl could trigger conformational changes and attractive diffusion interactions of ASN monomers towards the formation of mesoscopic assemblies with ordered internal structures and high thermostabilities. We discovered that pulsed EFs and ionic strength can modulate effectively the thermostability and autofluorescence effect of mesoscopic ASN assemblies by tuning the molecular interaction and arrangement. Remarkably, a specie of thermostable ASN assemblies showing a maximum autofluorescence emission at approx. 700 nm was synthesized applying 250 mM NaCl and the distinct pulsed EF, which could be attributed to the increase of β-sheet structures and hydrogen-bond networks within ASN assemblies. In summary, the presented data provide novel insights for modulating the growth kinetics, structures, and physicochemical properties of bio-macromolecular mesoscopic assemblies.