Achieving secondary structural resolution in kinetic measurements of protein folding: a case study of the folding mechanism of Trp-cage.
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ABSTRACT:
SUBMITTER: Culik RM
PROVIDER: S-EPMC3331591 | biostudies-literature | 2011 Nov
REPOSITORIES: biostudies-literature
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Achieving secondary structural resolution in kinetic measurements of protein folding: a case study of the folding mechanism of Trp-cage.
Culik Robert M RM Serrano Arnaldo L AL Bunagan Michelle R MR Gai Feng F
Angewandte Chemie (International ed. in English) 20110929 46
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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.
| S-EPMC2711228 | biostudies-literature
Project description:We develop a coarse-grained protein model with a simplified amino acid interaction potential. Using this model, we perform discrete molecular dynamics folding simulations of a small 20-residue protein--Trp-cage--from a fully extended conformation. We demonstrate the ability of the Trp-cage model to consistently reach conformations within 2-angstroms backbone root-mean-square distance from the corresponding NMR structures. The minimum root-mean-square distance of Trp-cage conformations in simulations can be <1 angstroms. Our findings suggest that, at least in the case of Trp-cage, a detailed all-atom protein model with a molecular mechanics force field is not necessary to reach the native state of a protein. Our results also suggest that the success of folding Trp-cage in our simulations and in the reported all-atom molecular mechanics simulation studies may be mainly due to the special stabilizing features specific to this miniprotein.
| S-EPMC1304993 | biostudies-literature
Project description:Using the miniprotein Trp-cage as a model, we show that D-amino acids can be used to facilitate the delineation of protein folding mechanism. Specifically, we study the folding-unfolding kinetics of three Trp-cage mutants where the native glycine residue near the C-terminus of the α-helix is replaced by a D-amino acid. A previous study showed that these mutations increase the Trp-cage stability, due to a terminal capping effect. Our results show that the stabilizing effect of D-asparagine and D-glutamine originates almost exclusively from a decrease in the unfolding rate, while the D-alanine mutation results in a similar decrease in the unfolding rate, but it also increases the folding rate. Together, these results support a folding mechanism wherein the α-helix formation in the transition state is nucleated at the N-terminus, whereas those long-range native interactions stabilizing this helix are developed at the downhill side of the folding free energy barrier.
| S-EPMC3844134 | biostudies-literature
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.
| S-EPMC263783 | biostudies-literature
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.
| S-EPMC3707288 | biostudies-literature
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 μs 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.
| S-EPMC4179588 | biostudies-literature
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.
| S-EPMC164630 | biostudies-literature
Project description:We investigate the kinetic pathways of folding and unfolding of the designed miniprotein Trp- cage in explicit solvent. Straightforward molecular dynamics and replica exchange methods both have severe convergence problems, whereas transition path sampling allows us to sample unbiased dynamical pathways between folded and unfolded states and leads to deeper understanding of the mechanisms of (un)folding. In contrast to previous predictions employing an implicit solvent, we find that Trp-cage folds primarily (80% of the paths) via a pathway forming the tertiary contacts and the salt bridge, before helix formation. The remaining 20% of the paths occur in the opposite order, by first forming the helix. The transition states of the rate-limiting steps are solvated native-like structures. Water expulsion is found to be the last step upon folding for each route. Committor analysis suggests that the dynamics of the solvent is not part of the reaction coordinate. Nevertheless, during the transition, specific water molecules are strongly bound and can play a structural role in the folding.
| S-EPMC1595309 | biostudies-literature
Project description:In this work a new method for the automatic exploration and calculation of multidimensional free energy landscapes is proposed. Inspired by metadynamics, it uses several collective variables that are relevant for the investigated process and a bias potential that discourages the sampling of already visited configurations. The latter potential allows escaping a local free energy minimum following the direction of slow motions. This is different from metadynamics in which there is no specific direction of the biasing force and the computational effort increases significantly with the number of collective variables. The method is tested on the Ace-Ala3-Nme peptide, and then it is applied to investigate the Trp-cage folding mechanism. For this protein, within a few hundreds of nanoseconds, a broad range of conformations is explored, including nearly native ones, initiating the simulation from a completely unfolded conformation. Finally, several folding/unfolding trajectories give a systematic description of the Trp-cage folding pathways, leading to a unified view for the folding mechanisms of this protein. The proposed mechanism is consistent with NMR chemical shift data at increasing temperature and recent experimental observations pointing to a pivotal role of secondary structure elements in directing the folding process toward the native state.
| S-EPMC3817379 | biostudies-literature
Project description:We present a new approach to study a multitude of folding pathways and different folding mechanisms for the 20-residue mini-protein Trp-Cage using the combined power of replica exchange molecular dynamics (REMD) simulations for conformational sampling, transition path theory (TPT) for constructing folding pathways, and stochastic simulations for sampling the pathways in a high dimensional structure space. REMD simulations of Trp-Cage with 16 replicas at temperatures between 270 and 566 K are carried out with an all-atom force field (OPLSAA) and an implicit solvent model (AGBNP). The conformations sampled from all temperatures are collected. They form a discretized state space that can be used to model the folding process. The equilibrium population for each state at a target temperature can be calculated using the weighted-histogram-analysis method (WHAM). By connecting states with similar structures and creating edges satisfying detailed balance conditions, we construct a kinetic network that preserves the equilibrium population distribution of the state space. After defining the folded and unfolded macrostates, committor probabilities (P(fold)) are calculated by solving a set of linear equations for each node in the network and pathways are extracted together with their fluxes using the TPT algorithm. By clustering the pathways into folding "tubes", a more physically meaningful picture of the diversity of folding routes emerges. Stochastic simulations are carried out on the network, and a procedure is developed to project sampled trajectories onto the folding tubes. The fluxes through the folding tubes calculated from the stochastic trajectories are in good agreement with the corresponding values obtained from the TPT analysis. The temperature dependence of the ensemble of Trp-Cage folding pathways is investigated. Above the folding temperature, a large number of diverse folding pathways with comparable fluxes flood the energy landscape. At low temperature, however, the folding transition is dominated by only a few localized pathways.
| S-EPMC3059588 | biostudies-literature