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Mapping the structural changes of LCD-TDP43 during the liquid-liquid phase separation by different spectroscopic
Milad Amiri1, Mohammad Javad Masroor2, S Shirin Shahangian3
1Department of Biology, Faculty of Basic Sciences, University of Guilan, University Campus 2, Rasht, Iran.
Biophysical Chemistry
|September 7, 2025
Summary
Researchers developed a novel fluorescence method to study liquid-liquid phase separation (LLPS) in TDP-43, revealing a new intermediate state. This advance offers insights into neuropathogenesis and protein aggregation mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Understanding the molecular mechanisms of Liquid-Liquid Phase Separation (LLPS) in TAR DNA-binding protein 43 (TDP-43) is crucial for elucidating its role in neuropathogenesis.
- Hydrophobic interactions and aromatic residue stacking are key drivers of TDP-43 LLPS.
Purpose of the Study:
- To introduce a novel, probe-free excitation-emission matrix (EEM) fluorescence method for monitoring aromatic residue microenvironments and π-π stacking during TDP-43 LLPS.
- To characterize intermediate states and structural transitions within the TDP-43 LLPS pathway.
Main Methods:
- Excitation-emission matrix (EEM) fluorescence spectroscopy for real-time monitoring of aromatic residue interactions.
- Circular Dichroism (CD) and Fourier-Transform Infrared (FTIR) spectroscopy to identify protein secondary structures.
- Atomic Force Microscopy (AFM) and Thioflavin T (ThT) assay to confirm protein aggregation and fibrillation.
Main Results:
- The EEM fluorescence method successfully tracked microenvironmental changes and π-π stacking during LLPS.
- A novel intermediate state with an α-sheet structure was identified in the liquid droplet phase, distinct from previously reported structures.
- Hydrophobic clustering increased continuously during phase separation, with varying polarities observed.
- Distinct protein species, from monomers to amyloid fibrils, were observed, confirming TDP-43's amyloidogenic nature.
Conclusions:
- The developed 3D fluorescence method provides valuable insights into π-π interactions driving LLPS-dependent aggregation of intrinsically disordered proteins (IDPs) containing low-complexity domains (LCDs).
- The discovery of α-sheet non-fibrillar intermediates offers a new perspective on the aggregation mechanisms of LCD-containing proteins like TDP-43.
- This research advances the understanding of TDP-43's role in neurodegenerative diseases.
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