A Comprehensive Multidimensional-Embedded, One-Dimensional Reaction Coordinate for Protein Unfolding/Folding

Toofanny, Rudesh D.; Jönsson, Anna K.; Daggett, Valerie

doi:10.1016/j.bpj.2010.02.048

Cited by 26 publications

(43 citation statements)

References 36 publications

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“…An intermediate was detected in the simulations, both by reaction coordinate analysis of property space (23) (Fig. 3B, Right) and clustering the all-versus-all Cα rmsd matrix (24).…”

Section: Resultsmentioning

confidence: 94%

Malleability of folding intermediates in the homeodomain superfamily

Banachewicz

Religa

Schaeffer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation model for family members with an unstable HTH motif. The folding intermediate of EnHD has nearly native HTH structure, but it is not docked with helix1. The determinant of whether two-or three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed Φ-value analysis of the folding of the Pit1 homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helix1 was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Pit1 and EnHD: The isolated peptide corresponding to the HTH motif in Pit1 was not folded in the absence of H1. Independent molecular dynamics simulations of Pit1 unfolding found an intermediate with H1 misfolded onto the HTH motif. The Pit1 folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from two-to three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms.protein folding | temperature jump | diffusion collision | transition state T he nucleation-condensation mechanism was proposed from a Φ-value analysis of the folding of the protein chymotrypsin inhibitor 2 (CI2) (1, 2). The individual elements of secondary structure in CI2 are not independently stable, leading to highly cooperative formation of structure in the transition state (TS) for folding in which the helix is the best-formed region, stabilized by long-range interactions. No element of secondary structure is fully formed in the TS and its folding is kinetically two-state (3). It was hypothesized that multistate folding requires independently stable elements of structure or an unstable element of structure being stabilized by interacting with other elements. At the other extreme, the three-helix engrailed homeodomain, EnHD, is a small protein that folds ultrafast via a stable intermediate in line with the framework mechanism, whereby secondary structure forms first followed by docking of the helices (4, 5). Its folding intermediate consists of helix1 (H1) being helical but not docked on to the helix2-turn-helix3 (HTH) DNA binding motif (5, 6). The HTH motif is independently stable in the on-pathway intermediate (7). Studies of other members of the homoedomain superfamily show a slide from this multistate framework folding mechanism to two-state folding and nucleation condensation for members with less stable HTH motifs, consistent with our earlier hypoth...

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“…An intermediate was detected in the simulations, both by reaction coordinate analysis of property space (23) (Fig. 3B, Right) and clustering the all-versus-all Cα rmsd matrix (24).…”

Section: Resultsmentioning

confidence: 94%

Malleability of folding intermediates in the homeodomain superfamily

Banachewicz

Religa

Schaeffer

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…This suppression of internal protein dynamics also complies with experimental observations. 50 Effect of trehalose on the free energy landscape constructed by the property space approach Beck et al 43 and others 47,48,51 suggested that the construction of a reaction coordinate using one or two properties to quantify the extent of folding is not justified and requires examination of property space wherein each property provides significant discriminatory power between native and non-native conformations. In this context, we validated our previous inferences by constructing a property space composed of eight most informative properties viz.…”

Section: Resultsmentioning

confidence: 99%

Revisiting the conundrum of trehalose stabilization

Katyal

Deep

2014

Phys. Chem. Chem. Phys.

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Protein aggregation and loss of protein's biological functionality are manifestations of protein instability. Cosolvents, in particular trehalose, are widely accepted antidotes against such destabilization. Although numerous theories have been promulgated in the literature with regard to its mechanism of stabilization, the present scenario is still elusive in view of the discrepancies existing in them. To this end, we have revisited the conundrum and attempted to rationalize the mechanism by conducting thorough investigation of the effect of trehalose on the native, partially unfolded and denatured states of protein "Lysozyme" by means of molecular dynamic (MD) simulations under different temperature and concentration regimes. Two-dimensional contour plots along with principal component analysis suggest that trehalose molecules offer on-pathway stabilization unaltering the principal direction of protein's motion, although it slows down protein dynamics so that the protein gets trapped in the homogeneous ensemble of conformations closer to the native state. Free energy landscape reveals higher population of native compared to intermediate and denatured states. Delphi results and calculation of the preferential interaction parameter demonstrate that this relative stabilization of the native state can be ascribed to be the consequence of favourable interactions of trehalose with side chains of certain loci on the protein surface encompassing polar flexible residues. Stability of protein results from the observed difference in binding affinity of trehalose for native and denatured states of protein. Our findings are at variance with the common conception of relative destabilization of the denatured state. Rather, we provide evidence for relative stabilization of the native state. This stabilization is due to interplay of protein-trehalose, water-trehalose, water-water, protein-water and trehalose-trehalose interactions.

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“…A total of 39 physical properties were monitored for all simulations to create an alternate description of the trajectory in property space, which can be very helpful for comparing different trajectories (7,(10)(11)(12) (Fig. S2).…”

Section: Methodsmentioning

confidence: 99%

“…SASA of Trp48, the fluorescence probe for folding, was included as well, as were the radius of gyration and end-toend distance. PC analysis was carried out on the resulting normalized 39-dimensional property space as described previously (11). N and I were defined for further analysis as the structures in the bins in the PC1 vs. PC2 histogram that had >40% of the maximum bin count for the four simulation sets (SM and TT at 25°C and 225°C).…”

Section: Methodsmentioning

confidence: 99%

“…Structures that are close together when plotted in property space, or a projection thereof, have similar structural properties. Such an approach was taken to develop a general, protein-independent property space to compare folding pathways of different proteins (10,11), and a tailored, protein-specific property space was used previously for EnHD (7,12). Here, 39 properties were included, and a principal component (PC) analysis was performed on the property space.…”

Section: Effect Of Intermolecular Interactions On Unfolding Pathway Omentioning

confidence: 99%

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Multimolecule test-tube simulations of protein unfolding and aggregation

McCully

Beck

Daggett

2012

Proc. Natl. Acad. Sci. U.S.A.

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Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/ folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein-water hydrogen bonds were also replaced with protein-protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein-protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.protein folding | protein dynamics T he folding pathway of the Engrailed homeodomain (EnHD) has been extensively characterized through a combination of experimental and computational techniques. EnHD is a threehelix bundle protein, with helices I and II packing antiparallel and helix III docking across them (Fig. 1). EnHD folds and unfolds on ultrafast timescales, which makes its folding pathway a good candidate to be studied by simulation. The structure of the transition state (TS) was first predicted by molecular dynamics (MD) simulations (1) and later validated by experimental techniques (2). A folding intermediate was identified by experiment (3) and structurally characterized by MD (4), and the MD-predicted structure was later validated by NMR (5). In addition, the protein adheres to the principle of microscopic reversibility in simulations at its melting temperature where unfolding and refolding occur in a single continuous trajectory (6). Similarly, when high-temperature unfolding simulations are quenched to folding permissive temperatures, the protein refolds by the reverse of unfolding (7). Despite the fact that the experimental techniques used provide ensemble measurements and the MD simulations are single-molecule (SM) in nature, the agreement has been very good, allowing for a much richer description of the folding/unfolding process of EnHD ...

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A Comprehensive Multidimensional-Embedded, One-Dimensional Reaction Coordinate for Protein Unfolding/Folding

Cited by 26 publications

References 36 publications

Malleability of folding intermediates in the homeodomain superfamily

Malleability of folding intermediates in the homeodomain superfamily

Revisiting the conundrum of trehalose stabilization

Multimolecule test-tube simulations of protein unfolding and aggregation

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