Tryptophan Probes of TDP-43 C-Terminal Domain Amyloid Formation

Abstract

Aggregated TAR DNA-binding protein 43 (TDP-43) forms the cytoplasmic hallmarks associated with patients suffering from amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin. Under normal conditions, TDP-43 is a 414-amino acid protein; however, aggregates are enriched with N-terminal truncations which contain residues 267–414, known as the C-terminal domain of TDP-43 (TDP-43CTD). To gain residue-specific information on the aggregation process of TDP-43CTD, we created three single-Trp containing mutants (W385F/W412F, W334F/W412F, and W334F/W385F) by substituting two of the three native Trp residues with Phe, yielding fluorescent probes at W334, W385, and W412, respectively. Aggregation kinetics, secondary structure, and fibril morphology were compared to the wild-type protein using thioflavin-T fluorescence, Raman spectroscopy, and transmission electron microscopy, respectively. While only W334 is determined to be in the proteinase-K resistant core, all three sites are sensitive reporters of aggregation, revealing site-specific differences. Interestingly, W334 exhibited unusual multistep Trp kinetics, pinpointing a distinctive role for W334 and its nearby region during aggregation. This behavior is retained even upon seeding, suggesting the observed spectral change is related to fibril growth. This work provides new insights into the aggregation mechanism of TDP-43CTD and exemplifies the advantages of Trp as a site-specific environmentally sensitive fluorescent probe.

Watching liquid droplets of TDP-43CTD age by Raman spectroscopy

“…We note that these filaments at 4 h are not associated with droplets and appear similar to the intermediate fibrils previously observed in non–phase-separating conditions ( i.e. , no salt) for TDP-43 CTD ( 8 ). This led us to question whether the aggregation process that leads to large fibril bundles visible in the bright-field and confocal fluorescence images is in fact distinct from droplet solidification.…”

“…S2 and Table S3 ). The presence of α-helical conformation is unexpected as TDP-43 CTD aggregates are characterized to be β-sheet rich ( 8 , 20 ). The stabilization of the polypeptide structure inside the droplets by 4 h is also intriguing as this corresponds to the lag phase of aggregation.…”

“…Trp-to-Phe mutations (residues 334, 385, and 412) were done via a QuikChange site-directed mutagenesis kit (Agilent) using the following primers: 5′-GCAAGAGCTCCGGTTTTGGCATGT AACTCG-3′ (W412F), 5′-GGTGCGGCAATCGGCTTTGGTAGCGCAAGCAATG-3′ (W385F), and 5′-GCGCTGCAGT CTAGCTTTGGTATGATGGGCATG-3′ (W334F). WT or W334F/W385F/W412F (W free ) TDP-43 CTD plasmid was transformed into Escherichia coli BL21 (DE3) (Invitrogen), expressed and purified as previously described ( 8 ). TEV-cleaved proteins contain an N-terminal overhang consisting of residues GHM and were assessed with >95% purity by SDS-PAGE and LC–MS.…”

“…Furthermore, TAR DNA-binding protein 43 (TDP-43) and fused in sarcoma protein, proteins associated with phase-separated compartments ( e.g. , stress granules) in cells ( 6 , 7 ), also have amyloid-forming domains ( 7 , 8 ). The relationship between phase separation, protein aggregation, and disease remains to be elucidated.…”

“…Here, we investigated conformational changes of the C-terminal domain (CTD) of TDP-43 (TDP-43 CTD ) during the aging process of droplets with spatial resolution by pairing a Raman spectrometer with an inverted microscope. TDP-43 CTD was chosen because its phase separation and aggregation into amyloid fibrils have been established ( 8 , 11 , 12 ). Specifically, a TDP-43 CTD mutant (W334F/W385F/W412F, referred to as W free ) was used because of its improved solubility and purification yield ( Fig.…”