Fig. 3: HDX-MS reveals different conformations of D121A and pS129 compared to WT aSyn.

Bars represent differences in deuterium uptake of peptides along the sequence of differently compared aSyn variants (e.g. WT vs D121A aSyn) with the N-terminus region in blue, the NAC region in yellow, and the C-terminus of aSyn in red. Negative values represent increased deuterium uptake in the variant (a, b) or in the calcium bound state (c–e), correlating to more solvent exposure, and less hydrogen bonding. The start and end of each peptide is marked on the x-axis (see aSyn peptide map in Supplementary Fig. 11). Peptides containing the mutation were not comparable to WT aSyn and were removed from the data set, indicated by blank regions. Comparison of the deuterium uptake (in Dalton -Da) between a WT and D121A aSyn and b WT and pS129 aSyn showed no significant differences to WT aSyn. c In the presence of calcium, WT aSyn becomes significantly more deprotected (more solvent exposed/less hydrogen bonded) at the N-terminus and the NAC region, and at the same time becomes solvent protected at the C-terminus. d D121A aSyn is significantly more deprotected at the N-terminus and the NAC region upon calcium addition and solvent protected at the C-terminus. e pS129 aSyn is deprotected at the NAC region upon calcium addition and solvent protected at the C-terminus, while no significant differences were observed at the N-terminus. The grey dashed line signifies the error (1 s.d.) of six replicates collected per condition. Data acquired at each peptide were subjected to an unpaired Student’s t-test with alpha set to 1%. Each row was analysed individually, without assuming a consistent SD, individual two-tailed p values are presented in Supplementary Tables 1–5 and significant differences where p-values are ≤0.01 are presented by a *. Individual replicate values for deuterium uptake are presented in Supplementary Figs. 22–24.