The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein in human brain neurons and extracellular tissue is a hallmark of Parkinson disease. Although the stable protein β-structure in α-synuclein fibrils has been determined at high resolution, the status of the dynamically disordered domains and their responses to in vivo confinement and small-molecule interactions remain elusive. We use electron paramagnetic resonance spectroscopy of the spin probe, TEMPOL, colocalized with α-synuclein fibrils, and temperature-controlled, ice boundary confinement to characterize the physical and mechanical properties of the dynamically disordered protein and surrounding coupled solvent regions. The spin probe rotational correlation time at all temperature (T) values is characterized by two motional components, which are assigned to dynamically disordered protein terminal and hydration phases around the central fibril β-structure. The observed transformation from fast to slow motion components with decreasing T and increasing confinement is proposed to proceed through compaction 1 (protein collapse and folding) and compaction 2 (collapse and restriction) processes, intervened by tertiary structure consolidation and stable hydration layer formation. Hysteresis in the return, increasing T direction originates from frustration of cooperative dynamics of the spatially overlapping terminal domains and reveals bistability of compact, low-enthalpy and disordered high-entropy structures. Arrhenius and van't Hoff analyses support the proposed molecular model and indicate that compaction transitions are accessible under physiological conditions. Confinement relief effects of the small molecule dimethylsulfoxide are consistent with the model. The system and results provide a robust platform for assessment of α-synuclein fibril-targeted drugs.

Homeodomain transcription factors (TFs) recognize their DNA targets through both sequence-specific base contacts and readout of local DNA shape. Although intrinsic DNA structure is encoded by nucleotide sequence, it also undergoes protein-induced structural deformation upon binding. Yet, the interplay between intrinsic and protein-induced DNA shape remains unclear. Here, we dissect how these two readout modes determine binding specificity in a trimeric complex composed of the Drosophila Hox TF Sex combs reduced and its cofactors, Homothorax and Extradenticle. Guided by SELEX-seq data, we performed molecular dynamics simulations of this complex bound to sequences of varying binding affinities. We find that minor groove width reflects intrinsic DNA structure, whereas minor groove width fluctuations capture protein-induced stabilization and reshaping of the DNA. Within the trimeric complex, Homothorax reduces conformational fluctuations in an orientation- and sequence-dependent manner, with charged residues in its N-terminal arm playing key roles in DNA shape readout. This demonstrates that recognition involves a context-dependent balance between conformational selection and induced fit. We extend this analysis to two other homeodomain TFs, Distal-less and Engrailed, revealing that even closely related proteins produce distinct DNA shape signatures depending on sequence context. To translate these insights to novel sequences or mutant proteins, we evaluate whether AlphaFold 3 (AF3) can capture mutation-sensitive DNA shape readout. Although AF3 accurately reproduces wild-type structures, it struggles to predict how mutations or conformational dynamics alter DNA shape. To bridge this gap, we developed a hybrid pipeline integrating AlphaFold-based homology modeling, molecular dynamics simulations, and DeepPBS, a deep learning method for binding specificity prediction. This multiscale framework successfully captures the active modulation of DNA structure missed by AF3 alone, providing new insights into TF binding specificity and creating a roadmap for integrating deep learning and physics-based methods to study molecular mechanisms.