Coupling of Solvent and Backbone Dynamics in α-Synuclein Revealed by using Multi-Probe EPR Spectroscopy

𝛼-Synuclein (𝛼-syn) is an intrinsically disordered protein (IDP) involved in neural transmission and its dysfunction is a hallmark of Parkinson’s disease.  𝛼-Syn possesses context-sensitive structural plasticity that results in domain-specific, heterogeneous dynamics that connect structural disorder to function. Understanding the coupling between protein (backbone, surface) and surrounding solvent, and its sensitivity to confinement, is essential for describing 𝛼-syn behavior. Toward this, we use continuous-wave EPR spectroscopy and three nitroxide reporters: the free spin probe, TEMPOL, which senses protein-coupled solvent dynamics, and two site-directed spin labels, 4-maleimido-TEMPO (4MT) and dithione-based MTSL, covalently attached to Cys9 in 𝛼-syn (S9C). Temperature (T) is varied to control ice-boundary confinement (200–265 K).  TEMPOL tracking of protein hydration and dynamically disordered solvent components has revealed a three-stage, compaction 1/structure consolidation/compaction 2 response of 𝛼-syn to confinement.  4MT-S9C mobility is reduced overall, but consistent, and resolves low-confinement pre-organization of the N-terminus.  MTSL-S9C exhibits overall higher mobility, resolving tethered spin label motion from backbone dynamics that dictate 4MT-S9C motion.  The dual spin probe-label approach reveals how dynamic allostery among central and flanking domains shapes 𝛼-syn conformational behavior, establishing a platform for quantitative drug evaluation.