TAR DNA-binding protein of 43 kDa (TDP-43) is the primary component of intracellular, cytoplasmic aggregates found in the majority of Amyotrophic Lateral Sclerosis (ALS) cases and a large percentage of frontotemporal lobar degeneration with ubiquitin (FTLD-U) cases. The C-terminal domain (residues 267–414) of TDP-43 (TDP-43CTD) contains an overwhelming majority of ALS-linked TDP-43 mutations and is enriched in ALS aggregates. While it is well known that TDP-43CTD is important for aggregation in vivo, the molecular mechanisms of amyloid formation in vitro are not well understood. Here, we used native tryptophan residues of TDP-43CTD (W334, W385, and W412) to elucidate the in vitro aggregation process of TDP-43CTD by substituting two of the three Trp with Phe to create site-specific fluorescent probes. Thioflavin-T fluorescence monitored kinetics indicated minimal perturbations of the Trp-to-Phe mutations on aggregation, supported by indistinguishable β-sheet structure and fibril morphology as assessed by Raman spectroscopy and transmission electron microscopy, respectively. However, tryptophan fluorescence revealed site-specific spectral blue-shift and quenching upon amyloid formation for all sites, even the extremely C-terminal W412. Remarkably, W334 fluorescence exhibited a multi-step kinetics behavior that could not be bypassed by seeding with pre-formed fibrils, suggesting an obligatory local conformation transition that occurs during fibril growth. This work demonstrates the power of intrinsic fluorescent probes for understanding amyloid formation and informs on the mechanism of TDP-43CTD aggregation.

Site-specific tryptophan probes of TDP-43 C-terminal domain amyloid formation

ACS Spring Meeting 2021

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