Sampling the multiple folding mechanisms of Trp-cage in explicit solvent.
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ABSTRACT:
SUBMITTER: Juraszek J
PROVIDER: S-EPMC1595309 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Trp-cage is a 20-residue miniprotein, which is believed to be the fastest folder known so far. In this study, the folding free energy landscape of Trp-cage has been explored in explicit solvent by using an OPLSAA force field with periodic boundary condition. A highly parallel replica exchange molecular dynamics method is used for the conformation space sampling, with the help of a recently developed efficient molecular dynamics algorithm P3ME/RESPA (particle-particle particle-mesh Ewald/reference system propagator algorithm). A two-step folding mechanism is proposed that involves an intermediate state where two correctly formed partial hydrophobic cores are separated by an essential salt-bridge between residues Asp-9 and Arg-16 near the center of the peptide. This metastable intermediate state provides an explanation for the superfast folding process. The free energy landscape is found to be rugged at low temperatures, and then becomes smooth and funnel-like above 340 K. The lowest free energy structure at 300 K is only 1.50 A Calpha-RMSD (Calpha-rms deviation) from the NMR structures. The simulated nuclear Overhauser effect pair distances are in excellent agreement with the raw NMR data. The temperature dependence of the Trp-cage population, however, is found to be significantly different from experiment, with a much higher melting transition temperature above 400 K (experimental 315 K), indicating that the current force fields, parameterized at room temperature, need to be improved to correctly predict the temperature dependence.
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Project description:Energetics was analyzed for Trp-cage miniprotein in water to elucidate the solvation effect in heat denaturation. The solvation free energy was computed for a set of protein structures at room and high temperatures with all-atom molecular dynamics simulation combined with the solution theory in the energy representation, and its correlations were investigated against the intramolecular (structural) energy of the protein and the average interaction energy of the protein with the solvent water. It was observed both at room and high temperatures that the solvation free energy is anticorrelated to the structural energy and varies in parallel to the electrostatic component of the protein-water interaction energy without correlations to the van der Waals and excluded-volume components. When the set of folded structures sampled at room temperature was compared with the set of unfolded ones at high temperature, it was found that the preference order of the two sets is in correspondence to the van der Waals and excluded-volume components in the sum of the protein intramolecular and protein-water intermolecular interactions and is not distinguished by the electrostatic component.
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Project description:We report rate constant calculations and a reaction coordinate analysis of the rate-limiting folding and unfolding process of the Trp-cage mini-protein in explicit solvent using transition interface sampling. Previous transition path sampling simulations revealed that in this (un)folding process the protein maintains its compact configuration, while a (de)increase of secondary structure is observed. The calculated folding rate agrees reasonably with experiment, while the unfolding rate is 10 times higher. We discuss possible origins for this mismatch. We recomputed the rates with the forward flux sampling method, and found a discrepancy of four orders of magnitude, probably caused by the method's higher sensitivity to the choice of order parameter with respect to transition interface sampling. Finally, we used the previously computed transition path-sampling ensemble to screen combinations of many order parameters for the best model of the reaction coordinate by employing likelihood maximization. We found that a combination of the root mean-square deviation of the helix and of the entire protein was, of the set of tried order parameters, the one that best describes the reaction coordination.
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Project description:We report a single-copy tempering method for simulating large complex systems. In a generalized ensemble, the method uses runtime estimate of the thermal average energy computed from a novel integral identity to guide a continuous temperature-space random walk. We first validated the method in a two-dimensional Ising model and a Lennard-Jones liquid system. It was then applied to folding of three small proteins, trpzip2, trp-cage, and villin headpiece in explicit solvent. Within 0.5-1 microsecond, all three systems were reversibly folded into atomic accuracy: the alpha carbon root mean square deviations of the best folded conformations from the native states were 0.2, 0.4, and 0.4 A, for trpzip2, trp-cage, and villin headpiece, respectively.
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Project description:Trp-cage is a designed 20-residue polypeptide that, in spite of its size, shares several features with larger globular proteins.Although the system has been intensively investigated experimentally and theoretically, its folding mechanism is not yet fully understood. Indeed, some experiments suggest a two-state behavior, while others point to the presence of intermediates. In this work we show that the results of a bias-exchange metadynamics simulation can be used for constructing a detailed thermodynamic and kinetic model of the system. The model, although constructed from a biased simulation, has a quality similar to those extracted from the analysis of long unbiased molecular dynamics trajectories. This is demonstrated by a careful benchmark of the approach on a smaller system, the solvated Ace-Ala3-Nme peptide. For theTrp-cage folding, the model predicts that the relaxation time of 3100 ns observed experimentally is due to the presence of a compact molten globule-like conformation. This state has an occupancy of only 3% at 300 K, but acts as a kinetic trap.Instead, non-compact structures relax to the folded state on the sub-microsecond timescale. The model also predicts the presence of a state at Calpha-RMSD of 4.4 A from the NMR structure in which the Trp strongly interacts with Pro12. This state can explain the abnormal temperature dependence of the Pro12-delta3 and Gly11-alpha3 chemical shifts. The structures of the two most stable misfolded intermediates are in agreement with NMR experiments on the unfolded protein. Our work shows that, using biased molecular dynamics trajectories, it is possible to construct a model describing in detail the Trp-cage folding kinetics and thermodynamics in agreement with experimental data.
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Project description:We report a numerical study of the (un)folding routes of the truncated FBP28 WW domain at ambient conditions using a combination of four advanced rare event molecular simulation techniques. We explore the free energy landscape of the native state, the unfolded state, and possible intermediates, with replica exchange molecular dynamics. Subsequent application of bias-exchange metadynamics yields three tentative unfolding pathways at room temperature. Using these paths to initiate a transition path sampling simulation reveals the existence of two major folding routes, differing in the formation order of the two main hairpins, and in hydrophobic side-chain interactions. Having established that the hairpin strand separation distances can act as reasonable reaction coordinates, we employ metadynamics to compute the unfolding barriers and find that the barrier with the lowest free energy corresponds with the most likely pathway found by transition path sampling. The unfolding barrier at 300 K is approximately 17 k(B)T approximately 42 kJ/mol, in agreement with the experimental unfolding rate constant. This work shows that combining several powerful simulation techniques provides a more complete understanding of the kinetic mechanism of protein folding.
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Project description:Using alternate measures of fold stability for a wide variety of Trp-cage mutants has raised the possibility that prior dynamics T-jump measures may not be reporting on complete cage formation for some species. NMR relaxation studies using probes that only achieve large chemical shift difference from unfolded values on complete cage formation indicate slower folding in some but not all cases. Fourteen species have been examined, with cage formation time constants (1/kF) ranging from 0.9-7.5 ?s at 300 K. The present study does not change the status of the Trp-cage as a fast folding, essentially two-state system, although it does alter the stage at which this description applies. A diversity of prestructuring events, depending on the specific analogue examined, may appear in the folding scenario, but in all cases, formation of the N-terminal helix is complete either at or before the cage-formation transition state. In contrast, the fold-stabilizing H-bonding interactions of the buried Ser14 side chain and the Arg/Asp salt bridge are post-transition state features on the folding pathway. The study has also found instances in which a [P12W] mutation is fold destabilizing but still serves to accelerate the folding process.
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Project description:Using action-derived molecular dynamics (ADMD), we study the dynamic folding pathway models of the Trp-cage protein by providing its sequential conformational changes from its initial disordered structure to the final native structure at atomic details. We find that the numbers of native contacts and native hydrogen bonds are highly correlated, implying that the native structure of Trp-cage is achieved through the concurrent formations of native contacts and native hydrogen bonds. In early stage, an unfolded state appears with partially formed native contacts (~40%) and native hydrogen bonds (~30%). Afterward, the folding is initiated by the contact of the side chain of Tyr3 with that of Trp6, together with the formation of the N-terminal ? -helix. Then, the C-terminal polyproline structure docks onto the Trp6 and Tyr3 rings, resulting in the formations of the hydrophobic core of Trp-cage and its near-native state. Finally, the slow adjustment processes of the near-native states into the native structure are dominant in later stage. The ADMD results are in agreement with those of the experimental folding studies on Trp-cage and consistent with most of other computational studies.
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Project description:Replica-exchange molecular dynamics simulations in implicit solvent have been carried out to study the folding thermodynamics of a designed 20-residue peptide, or "miniprotein." The simulations in this study used the amber (parm94) force field along with the generalized Born/solvent-accessible surface area implicit solvent model, and they spanned a range of temperatures from 273 to 630 K. Starting from a completely extended initial conformation, simulations of one peptide sequence sample conformations that are <1.0 A Calpha rms positional deviation from structures in the corresponding NMR ensemble. These folded states are thermodynamically stable with a simulated melting temperature of approximately 400 K, and they satisfy the majority of experimentally observed NMR restraints. Simulations of a related mutant peptide show a degenerate ensemble of states at low temperature, in agreement with experimental results.
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Project description:We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent using the Amberff99SB force field. Replica-exchange molecular dynamics simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states at two densities. The obtained ensembles are shown to reach equilibrium in the 1 mus/replica timescale. The total simulation time used in the calculations exceeds 100 mus. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, DeltaG(P, T), over the whole region of temperatures and pressures sampled in the simulations. The DeltaG(P, T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure-induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (T(f) = 321 K), free energy, and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semiempirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins.
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