Dataset Information

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

ABSTRACT: 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 (I_N and I_U) 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 I_N 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.

SUBMITTER: Prakash A

PROVIDER: S-EPMC9088055 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

Prakash Amresh A Kumar Vijay V Meena Naveen Kumar NK Lynn Andrew M AM

RSC advances 20180530 35

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 ...[more]

PMID: 35548664

Similar Datasets

Project description: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.

Project description:BackgroundFrontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain.MethodsWe have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies.ResultsThe fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (< 1 min) round, homogeneous and 0.5-1.0 µm wide assemblies which then coalesced into larger, yet round, species. When labelled with AlexaFluor488, they initially exhibited fluorescence recovery after photobleaching (FRAP), showing a liquid behaviour distinct from full-length TDP-43 and similar to PrLD. The protein molecules did not undergo major structural changes, as determined with circular dichroism and intrinsic fluorescence spectroscopies. This process had a pH and salt dependence distinct from those of full-length TDP-43 and its PrLD, which can be rationalized on the grounds of electrostatic forces.ConclusionsSimilarly to PrLD, PrLD-less TDP-43 forms liquid droplets in vitro through liquid-liquid phase separation (LLPS), unlike the full-length protein that rather undergoes liquid-solid phase separation (LSPS). These results offer a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. On the one hand, PrLD-less TDP-43 has a low pI and oppositively charged domains, and LLPS is inhibited by salts, which attenuate inter-domain electrostatic attractions. On the other hand, PrLD is positively charged due to a high isoionic point (pI) and LLPS is therefore promoted by salts and pH increases as they both reduce electrostatic repulsions. By contrast, full-length TDP-43 undergoes LSPS most favourably at its pI, with positive and negative salt dependences at lower and higher pH, respectively, depending on whether repulsive or attractive forces dominate, respectively.

Dataset Information

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

Publications

Elucidation of the structural stability and dynamics of heterogeneous intermediate ensembles in unfolding pathway of the N-terminal domain of TDP-43.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets