Time-Resolved SAXS Reveals Distinct Millisecond Metal-Induced Conformational Dynamics of Monomeric α-Synuclein.

Transition metal ions have been implicated in modulation the conformational behavior and aggregation of WT α-synuclein (WT-αSyn), associated with Parkinson's disease pathology. Nevertheless, the initial structural rearrangements that drive aggregation are not fully understood. Here, we employed time-resolved small-angle X-ray scattering (TR-SAXS) in a microfluidic setup to investigate the structural dynamics of monomeric WT-αSyn upon interaction with Mn2 +, Fe3 +, Cu2 +, and Zn2 +. Using Guinier analysis, GNOM, and Ensemble Optimization Method (EOM), we resolved distinct, metal-specific conformational transitions on the sub-second timescale. Fe3 + induced rapid and sustained compaction of αSyn, while Cu2 + promoted extended and heterogeneous conformations, expanding the C-terminal domain, and disrupting global folding. In contrast, Mn2 + and Zn2 + led to more gradual, domain-specific compaction. Fractal dimension analysis and hierarchical clustering further revealed Fe3 + and Zn2 + enriched compaction states, while Cu2 + favored intermediate species potentially linked to early aggregation. These findings highlight how metal ion binding differentially and initially reshape the conformation ensemble of WT-αSyn, offering mechanistic insight into metal-induced misfolding pathways relevant to synucleinopathies.