A Complex Equilibrium among Partially Unfolded Conformations in Monomeric Transthyretin

Conti, Simona; Li, Xinyi; Gianni, Stefano; Ghadami, Seyyed Abolghasem; Buxbaum, Joel N.; Cecchi, Cristina; Chiti, Fabrizio; Bemporad, Francesco

doi:10.1021/bi500430w

Cited by 12 publications

(14 citation statements)

References 62 publications

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“…It is also worth noting that S0, which most strongly resembles the native structure, has the lowest equilibrium probability (about 2.40%), suggesting the instability for this near native state. By contrast, S4, with the largest RMSDs compared to native structure, has the largest population (about 85.28%), which means that this partially unfolded state is extremely stable and may serve as an aggregation-prone state for further TTR aggregation, consistent with the findings of Conti et al [57]. Therefore, taking the above-obtained results together, we then treated S0 as the native state, while treating S4 with partially unfolded conformations and the largest equilibrium probability as the potential misfolded state to further explore the potential misfolding mechanism of TTR monomer.…”

Section: Structural Ensemble Of Key States Of Transthyretin Monomer Msupporting

confidence: 89%

Exploration of the Misfolding Mechanism of Transthyretin Monomer: Insights from Hybrid-Resolution Simulations and Markov State Model Analysis

et al. 2019

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Misfolding and aggregation of transthyretin (TTR) is widely known to be responsible for a progressive systemic disorder called amyloid transthyretin (ATTR) amyloidosis. Studies suggest that TTR aggregation is initiated by a rate-limiting dissociation of the homo-tetramer into its monomers, which can rapidly misfold and self-assemble into amyloid fibril. Thus, exploring conformational change involved in TTR monomer misfolding is of vital importance for understanding the pathogenesis of ATTR amyloidosis. In this work, microsecond timescale hybrid-resolution molecular dynamics (MD) simulations combined with Markov state model (MSM) analysis were performed to investigate the misfolding mechanism of the TTR monomer. The results indicate that a macrostate with partially unfolded conformations may serve as the misfolded state of the TTR monomer. This misfolded state was extremely stable with a very large equilibrium probability of about 85.28%. With secondary structure analysis, we found the DAGH sheet in this state to be significantly destroyed. The CBEF sheet was relatively stable and sheet structure was maintained. However, the F-strand in this sheet was likely to move away from E-strand and reform a new β-sheet with the H-strand. This observation is consistent with experimental finding that F and H strands in the outer edge drive the misfolding of TTR. Finally, transition pathways from a near native state to this misfolded macrostate showed that the conformational transition can occur either through a native-like β-sheet intermediates or through partially unfolded intermediates, while the later appears to be the main pathway. As a whole, we identified a potential misfolded state of the TTR monomer and elucidated the misfolding pathway for its conformational transition. This work can provide a valuable theoretical basis for understanding of TTR aggregation and the pathogenesis of ATTR amyloidosis at the atomic level.

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Section: Structural Ensemble Of Key States Of Transthyretin Monomer Msupporting

confidence: 89%

Exploration of the Misfolding Mechanism of Transthyretin Monomer: Insights from Hybrid-Resolution Simulations and Markov State Model Analysis

et al. 2019

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“…Aggregates were also found in the samples containing M-TTR (Fig. 5C), where they completely mask the peak related to the soluble form of M-TTR found at 4.50.6 nm (Conti et al, 2014;Pires et al, 2012;Jiang et al, 2001). …”

Section: Resultsmentioning

confidence: 93%

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Cappelli

Penco²,

Mannini³

et al. 2016

Biological Chemistry

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. (2016). Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations. Biological Chemistry: official scientific journal of the GBM, 397 (5), 401-415. Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations AbstractLiving systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsCappelli, S., Penco, A., Mannini, B., Cascella, R., Wilson, M. R., Ecroyd, H., Li, X., Buxbaum, J. N., Dobson, C. M., Cecchi, C., Relini, A. & Chiti, F. (2016 measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasises the efficiency and versatility of these protein molecules.

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“…Classically, an effective strategy to discriminate on‐ versus off‐pathway intermediates was applied when there is kinetic coupling between a fast phase, mainly associated with the formation/breakage of the folding intermediate, and the slower phase, mainly reporting the accumulation/unfolding of the native state . Under such conditions, the pre‐equilibrium assumption breaks down and, in some cases, the on‐ and off‐pathway scenarios differ substantially.…”

Section: Resultsmentioning

confidence: 99%

Structural characterization of an on‐pathway intermediate and transition state in the folding of the N‐terminal SH2 domain from SHP2

et al. 2019

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Src Homology 2 (SH2) domains are a class of protein domains that present a conserved three‐dimensional structure and possess a crucial role in mediating protein‐protein interactions. Despite their importance and abundance in the proteome, knowledge about the folding properties of SH2 domain is limited. Here we present an extensive mutational analysis (Φ value analysis) of the folding pathway of the N‐SH2 domain of the Src homology region 2 domain‐containing phosphatase‐2 (SHP2) protein, a 104 residues domain that presents the classical SH2 domain fold (two α‐helices flanking a central β‐sheet composed of 3‐5 antiparallel β‐strands), with a fundamental role in mediating the interaction of SHP2 with its substrates and triggering key metabolic pathways in the cell. By analysing folding kinetic data we demonstrated that the folding pathway of N‐SH2 presents an obligatory on‐pathway intermediate that accumulates during the folding reaction. The production of 24 conservative site‐directed variants allowed us to perform a Φ value analysis, by which we could fully characterize the intermediate and the transition state native‐like interactions, providing a detailed quantitative analysis of the folding pathway of N‐SH2. Results highlight the presence of a hydrophobic nucleus that stabilizes the intermediate, leading to a higher degree of native‐like interactions in the transition state. Data are discussed and compared with previous works on SH2 domains.

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A Complex Equilibrium among Partially Unfolded Conformations in Monomeric Transthyretin

Cited by 12 publications

References 62 publications

Exploration of the Misfolding Mechanism of Transthyretin Monomer: Insights from Hybrid-Resolution Simulations and Markov State Model Analysis

Exploration of the Misfolding Mechanism of Transthyretin Monomer: Insights from Hybrid-Resolution Simulations and Markov State Model Analysis

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Structural characterization of an on‐pathway intermediate and transition state in the folding of the N‐terminal SH2 domain from SHP2

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