# Detection of an Intermediate in the Unfolding Process of the N-Terminal Domain of TDP-43

## TL;DR

The study identifies a partially unfolded intermediate state in the N-terminal domain of TDP-43 protein, which may be important for its function and disease-related behavior.

## Contribution

The novel contribution is the discovery of a distinct intermediate state during the unfolding of TDP-43's N-terminal domain.

## Key findings

- A partially unfolded intermediate state forms during TDP-43 NTD unfolding, detectable by fluorescence and H/D exchange.
- The intermediate has a distorted β-sheet and exposed hydrophobic regions, indicating structural changes.
- The intermediate forms independently of TDP-43 oligomeric state and is a partially folded dimer at high concentration.

## Abstract

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein
accumulating
in intraneuronal cytoplasmic inclusions associated with amyotrophic
lateral sclerosis, frontotemporal lobar degeneration with tau-negative/ubiquitin-positive
inclusions, and limbic-predominant age-related TDP-43 encephalopathy.
Oligomerization of full-length TDP-43, driven by its N-terminal domain
(NTD), is essential for its function, but aberrant self-assembly also
promotes liquid–liquid phase separation and formation of solid
inclusions. Building on recent all-atom molecular dynamics simulations
and using various biophysical approaches, we identified a partially
unfolded state accumulating during unfolding of TDP-43 NTD, before
the major energy barrier of unfolding is crossed. Intrinsic fluorescence
spectroscopy coupled to a stopped-flow device at high urea concentration
reveals that the intermediate state has a fluorescence emission distinct
from those of the native and unfolded states and forms within the
14 ms dead time. Conventional fluorescence spectroscopy shows it still
accumulates at moderate urea concentration. Circular dichroism and
H/D exchange results show a species with an intermediate content of
secondary structure and a distorted β-sheet, whereas SYPRO orange
fluorescence indicates an open conformation with more exposed hydrophobic
regions compared to the native state. Importantly, this intermediate
is observed even at low protein concentration, when TDP-43 NTD is
largely monomeric, indicating that its formation is independent of
the initial TDP-43 NTD oligomeric state. Dynamic light scattering
at high protein concentration shows that the intermediate is a partially
folded dimer. The intermediate forms upon chemical denaturation and
does not occur under thermal unfolding. Overall, the findings highlight
the presence of one more partially folded state for TDP-43 NTD, underlining
its high structural plasticity and suggesting that its distinct unfolding
pathway may play a critical role in both its functional and pathological
behaviors.