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

ABSTRACT 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 Mn 2 + , Fe 3 + , Cu 2 + , and Zn 2 + . Using Guinier analysis, GNOM, and Ensemble Optimization Method (EOM), we resolved distinct, metal‐specific conformational transitions on the sub‐second timescale. Fe 3 + induced rapid and sustained compaction of αSyn, while Cu 2 + promoted extended and heterogeneous conformations, expanding the C‐terminal domain, and disrupting global folding. In contrast, Mn 2 + and Zn 2 + led to more gradual, domain‐specific compaction. Fractal dimension analysis and hierarchical clustering further revealed Fe 3 + and Zn 2 + enriched compaction states, while Cu 2 + 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.