The N-terminal domain of the RNA binding protein TDP-43 (NTD) is essential to both physiology and proteinopathy; however, elucidation of its folding/unfolding still remains a major quest. In this study, we have investigated the biophysical behavior of intermediate ensembles employing all-atom molecular dynamics simulations in 8 M urea accelerated with high temperatures to achieve unfolded states in a confined computation time. The cumulative results of the 2.75 μs simulations show that unfolding of the NTD at 350 K evolves through different stable and meta-stable intermediate states. The free-energy landscape reveals two meta-stable intermediates (IN and IU) stabilized by non-native interactions, which are largely hydrophilic and highly energetically frustrated. A single buried tryptophan residue, W80, undergoes solvent exposure to different extents during unfolding; this suggests a structurally heterogeneous population of intermediate ensembles. Furthermore, the structure properties of the IN state show a resemblance to the molten globule (MG) state with most of the secondary structures intact. The unfolding of the NTD is initiated by the loss of β-strands, and the unfolded (U) states exhibit a population of non-native α-helices. These non-native unfolded intermediate ensembles may mediate protein oligomerization, leading to the formation of pathological, irreversible aggregates, characteristics of disease pathogenesis.
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