Mapping the structural changes of LCD-TDP43 during the liquid-liquid phase separation by different spectroscopic platforms

A comprehensive understanding of the molecular mechanism underlying the Liquid-Liquid Phase Separation (LLPS) pathway of LCD-TDP43 remains a challenge in the context of its neuropathogenesis. The primary driving force behind the TDP-43 LLPS is the interplay of hydrophobic interactions reinforced by aromatic residues. This study presents a novel, convenient, sensitive, and probe-free approach using excitation-emission matrix (EEM) fluorescence to monitor the microenvironment of aromatic residues and π-π stacking interactions during different stages of the LLPS pathway. Protein local structuring and the alterations in the positions of aromatic residues, individually and collectively, were detected by this life-time 3D fingerprinting. A new intermediate state with a unique α-sheet structure in the liquid droplet state and other transient species up to amyloid fibrils was discovered by CD and FTIR analyses. This structure with an inherent tendency for transition to β-amyloids, has not previously been reported in the context of LCD-TDP43 nor other LLPS-prone proteins. Mapping of hydrophobic clustering during phase separation revealed a continuous increase, accompanied by different surrounding polarities. The formation of distinct protein species within the LLPS pathway (from monomer to fibril), along with the amyloidogenic nature of TDP-43 fibrillation, was also confirmed by AFM analysis and ThT assay. To conclude, the 3D fluorescence method introduced in this study provides an effective and straightforward approach to critical valuable insights into the key π-π interactions in the LLPS-dependent aggregation pathway of LCD-containing IDPs. The novel identification of the α-sheet non-fibrilar intermediates may provide a new perspective for elucidating the aggregation mechanism of these proteins.