α-Synuclein (αSyn) is an intrinsically disordered protein implicated in the pathogenesis of Parkinson’s disease and related synucleinopathies through its propensity to misfold and aggregate into neurotoxic species. Transition metal ions such as Mn²⁺, Fe³⁺, Cu²⁺, and Zn²⁺ are enriched in the diseased brain and have been reported to modulate αSyn aggregation; however, their real-time impact on the conformational ensemble of monomeric αSyn remains incompletely understood.
In this study, we employed time-resolved small-angle X-ray scattering (TR-SAXS) using a microfluidic mixing device to capture the sub-second structural dynamics of WT-αSyn in response to transition metal binding. Structural changes were quantified using Guinier analysis, GNOM, fractal dimension analysis, and ensemble optimization modeling (EOM), providing a multidimensional view of the protein’s behavior. Our findings reveal distinct metal-specific effects. Fe³⁺ triggered rapid and sustained global compaction of αSyn, reducing the radius of gyration (Rg) and enriching compact conformers without detectable intermediates. In contrast, Cu²⁺ initially promoted extended, heterogeneous conformations and significantly expanded the C-terminal domain, suggesting formation of partially folded intermediates. Zn²⁺ and Mn²⁺ had more subtle, domain-specific effects, generally promoting modest compaction with minimal disruption of native dynamics. Metal ions not only influence global compaction but also reshape inter-domain interactions in unique ways. Together, these results provide mechanistic insight into how transition metal ions modulate the conformation landscape of αSyn on a millisecond timescale, with implications for early misfolding steps in neurodegeneration. Our approach demonstrates the power of TR-SAXS in resolving the real-time dynamics of intrinsically disordered protein and supports future therapeutic strategies targeting metal-induced misfolding pathways.

Miriam Santos Pinto

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