Extended Data Fig. 4: α-Syn nanoclusters are kinetically and thermodynamically less stable than oligomers.
From: Mass photometric detection and quantification of nanoscale α-synuclein phase separation
(Left) MWapp histograms obtained from mass photometry measurements of 60 μM α-Syn + 20% (w/v) PEG before (blue) and after (red) 2x dilution are shown. MWapp histograms of the same sample after addition of 1 M urea (orange) are also reported. Note that addition of 1 M urea leads to a final concentration of 50 μM α-Syn + 17% (w/v) PEG which is still in the nanocluster regime (Supplementary Fig. 6). Therefore, disappearance of clusters indicated urea-induced destabilization/dissolution. Representative mass photometry images (ratiometric) of the nanoclusters before and after dilution are shown. (Right) MWapp histograms obtained with 1 μM α-Syn oligomers, purified by size exclusion chromatography, before dilution (green), after 2x dilution (grey) and after addition of 1 M urea (orange). Representative mass photometry images (ratiometric) of the oligomers before dilution, after dilution and after addition of 1 M urea are shown. All the experiments are performed in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 and at 25 °C. The observed persistence of oligomers even after urea treatment is in stark contrast to the dynamic nature of the nanoclusters we describe here and therefore we believe that the clusters do genuinely represent a hitherto unseen assembly state of α-Syn. The experiments are performed n = 2 independent times with similar results. PEG: PEG-8000 unless mentioned otherwise.
