Proteoform-Resolved Cross-Linking Reveals Environment-Dependent Structural Effects of α-Synuclein S129 Phosphorylation.

Intrinsically disordered proteins (IDPs) drive many neurodegenerative disorders, but their structures remain difficult to define because they populate dynamic ensembles that change with the chemical environment. This problem is central for α-synuclein (aSyn), a Parkinson's disease-linked IDP in which S129 phosphorylation is highly enriched in disease-associated aggregates. Although S129 phosphorylation stabilizes a more compact aSyn ensemble in dilute solution, whether this structural effect persists across other biochemical environments has remained unknown. Here, we developed a quantitative cross-linking mass spectrometry framework to determine how WT and pS129 aSyn respond to chemically distinct environments. We compared dilute buffer with two perturbations relevant to aSyn biology: trimethylamine N-oxide (TMAO), a gut-microbiome-derived metabolite associated with Parkinson's disease that can also act as a compacting osmolyte at high concentration, and octyl glucoside (OG) micelles, which provide a membrane-mimetic surface. TMAO rewired the phosphorylation-dependent structural response in a concentration-dependent manner: 1.8 M TMAO shifted WT aSyn toward the dilute pS129 contact pattern by increasing long-range contacts between the N-terminal and C-terminal regions, whereas 3.4 M TMAO collapsed both proteoforms and reduced their structural differences. In OG micelles, the proteoforms diverged. WT favored an extended-helix-like contact pattern with stronger contacts between the C-terminal tail and micelle-bound N-terminal region, whereas pS129 favored contacts between the N-terminal and NAC regions and fewer contacts to the C-terminal tail, consistent with a broken-helix-like topology. By integrating regional contact counts, normalized cross-link intensities, and geometric compatibility analysis, this workflow distinguishes broad contact accessibility from the residue-level contacts that dominate each condition. These results establish a proteoform-by-environment model for IDP structure, in which phosphorylation effects are not fixed but are rewritten by the surrounding chemical environment.