Fig. 2. ATP elicits a concentration-dependent effect on αS aggregation that is phosphate-driven and is preserved in the presence of magnesium. (a) ThT fluorescence of fresh 300 μM WT αS in ThT buffer (20 mM K₂HPO₄, 5 mM KH₂PO₄, 100 mM KCl, 200 μM EDTA, 0.05% NaN₃), incubated for 85 h in a 37 °C plate reader. Plotted are average ThT measurements across four wells per condition, with measurements taken every six minutes with 30 s orbital shaking prior to each read and normalized to the final measurement for the αS sample. Inset shows non-normalized data, with standard deviations for the αS and αS + 10 mM ATP conditions shown. Error bars for ATP < 10 mM are not shown to avoid overcrowding, but are comparable to 10 mM ATP. (b) ThT fluorescence of fresh 300 μM WT αS in ThT buffer, incubated for 72 h in a 37 °C shaker at 150 rpm then in a plate reader for 20 additional hours at plateau. Plotted are well-specific average ThT measurements from multiple independent experiments, taken at plateau every five min with 30 s orbital shaking prior to each read and normalized to the average measurement of all αS samples. Ligand concentrations are all 10 mM. Chemical structures of AMP, ADP, ATP and triphosphate at pH 7.4 are shown above the panel. (c) ThT fluorescence of fresh 440 μM WT αS in ThT buffer, incubated for 47 h in a 37 °C plate reader. Plotted are average ThT measurements across four wells per condition, with measurements taken every six minutes with 30 s orbital shaking prior to each read and normalized to the final measurement of the αS + 10 mM MgCl₂ sample. (d) Well-specific average ThT fluorescence measurements of panel (c) samples at plateau (40–47 h), normalized to the average measurement of all αS + 10 mM MgCl₂ samples. Straight lines between samples in panels (b) and (d) represent no significant difference. Sample comparisons in panels (b) and (d) represent significance levels: * = p < 0.05, *** = p < 0.001 and **** = p < 0.0001.