Residue-Resolved Mapping of Heterogeneous Structural Organization during Native-to-Amyloid Transition of an...

Protein misfolding and aggregation in neuronal cells are key molecular events underlying several fatal neurodegenerative disorders. Effective therapeutic intervention requires residue-resolved structural information on the early aggregation intermediates that precede amyloid formation. However, these transient and heterogeneous species remain poorly characterized because of their dynamic nature and the limitations of conventional structural approaches. Here, we employ a multisite fluorescence approach to obtain residue-specific insights into the misfolding and aggregation pathway of the nucleic acid-binding domain of TDP-43. Using a library of single-tryptophan-single-cysteine mutants combined with site-selective labeling, we systematically probe intramolecular distances, local solvation, conformational rigidity, and structural fluctuations across native, molten globule, oligomeric, and amyloid states. The results reveal that the early amyloidogenic molten globule retains native-like secondary structure but undergoes heterogeneous structural reorganization, including simultaneous local expansion and compaction, as revealed by multisite Förster resonance energy transfer. Residue-specific analyses also identify early unfolding sites and regions that progressively become incorporated into the amyloid core. These findings provide a residue-resolved view of the structural transitions that link native conformations to aggregation-prone states and establish a framework for understanding early events in TDP-43 misfolding.