Ab initio folding of proteins with all-atom discrete molecular dynamics

Discrete molecular dynamics (DMD) is a rapid sampling method used in protein folding and aggregation studies. Until now, DMD was used to perform simulations of simplified protein models in conjunction with structure-based force fields. Here, we develop an all-atom protein model and a transferable force field featuring packing, solvation, and environment-dependent hydrogen bond interactions. We performed folding simulations of six small proteins (20-60 residues) with distinct native structures by the replica exchange method. In all cases, native or near-native states were reached in simulations. For three small proteins, multiple folding transitions are observed, and the computationally characterized thermodynamics are in qualitative agreement with experiments. The predictive power of all-atom DMD highlights the importance of environment-dependent hydrogen bond interactions in modeling protein folding. The developed approach can be used for accurate and rapid sampling of conformational spaces of proteins and protein-protein complexes and applied to protein engineering and design of protein-protein interactions.     

Ab initio RNA folding by discrete molecular dynamics: from structure prediction to folding mechanisms

RNA molecules with novel functions have revived interest in the accurate prediction of RNA three-dimensional (3D) structure and folding dynamics. However, existing methods are inefficient in automated 3D structure prediction. Here, we report a robust computational approach for rapid folding of RNA molecules. We develop a simplified RNA model for discrete molecular dynamics (DMD) simulations, incorporating base-pairing and base-stacking interactions. We demonstrate correct folding of 150 structurally diverse RNA sequences. The majority of DMD-predicted 3D structures have <4 A deviations from experimental structures. The secondary structures corresponding to the predicted 3D structures consist of 94% native base-pair interactions. Folding thermodynamics and kinetics of tRNA(Phe), pseudoknots, and mRNA fragments in DMD simulations are in agreement with previous experimental findings. Folding of RNA molecules features transient, non-native conformations, suggesting non-hierarchical RNA folding. Our method allows rapid conformational sampling of RNA folding, with computational time increasing linearly with RNA length. We envision this approach as a promising tool for RNA structural and functional analyses.     

Assembly of a tetrameric alpha-helical bundle: computer simulations on an intermediate-resolution protein model.

Discontinuous molecular dynamics (DMD) simulation on an intermediate-resolution protein model is used to study the folding of an isolated, small model peptide to an amphipathic alpha-helix and the assembly of four of these model peptides into a four-helix bundle. A total of 129 simulations were performed on the isolated peptide, and 50 simulations were performed on the four-peptide system. Simulations efficiently sample conformational space allowing complete folding trajectories from random initial configurations to be observed within 15 min for the one-peptide system and within 15 h for the four-peptide system on a 500-MHz workstation. The native structures of both the alpha-helix and the four-helix bundle are consistent with experimental characterization studies and with results from previous simulations on these model peptides. In both the one- and four-peptide systems, the native state is achieved during simulations within an optimal temperature range, a phenomenon also observed experimentally. The ease with which our simulations yield reasonable estimates of folded structures demonstrates the power of the intermediate-resolution model developed for this work and the DMD algorithm and suggests that simulations of very long times and of multiprotein systems may be possible with this model.     

New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate

Mounting evidence suggests that the focal adhesion targeting (FAT) domain, an antiparallel four-helix bundle, exists in alternative conformations that may modulate phosphorylation, ligand binding, and the subcellular localization of focal adhesion kinase (FAK). In order to characterize the conformational dynamics of the FAT domain, we have developed a novel method for reconstructing the folding pathway of the FAT domain by using discrete molecular dynamics (DMD) simulations, with free energy constraints derived from NMR hydrogen exchange data. The DMD simulations detect a folding intermediate, in which a cooperative unfolding event causes helix 1 to lose helical character while separating from the helix bundle. The conformational dynamic features of helix 1 in the intermediate state of the FAT domain are likely to facilitate Y926 phosphorylation, yet interfere with paxillin binding. The presence of this intermediate state in vivo may promote FAK signaling via the ERK/MAPK pathway and by release of FAK from focal adhesions.     

A hidden aggregation‐prone structure in the heart of hypoxia inducible factor prolyl hydroxylase

Prolyl hydroxylase domain‐containing protein 2 (PHD2), as one of the most important regulators of angiogenesis and metastasis of cancer cells, is a promising target for cancer therapy drug design. Progressive studies imply that abnormality in PHD2 function may be due to misfolding. Therefore, study of the PHD2 unfolding pathway paves the way for a better understanding of the influence of PHD2 mutations and cancer cell metabolites on the protein folding pathway. We study the unfolding of the PHD2 catalytic domain using differential scanning calorimetry (DSC), fluorescence spectroscopy, and discrete molecular dynamics simulations (DMD). Using computational and experimental techniques, we find that PHD2 undergoes four transitions along the thermal unfolding pathway. To illustrate PHD2 unfolding events in atomic detail, we utilize DMD simulations. Analysis of computational results indicates an intermediate species in the PHD2 unfolding pathway that may enhance aggregation propensity, explaining mutation‐independent PHD2 malfunction. Proteins 2016; 84:611–623. © 2016 Wiley Periodicals, Inc.     

Ten-microsecond molecular dynamics simulation of a fast-folding WW domain

All-atom molecular dynamics (MD) simulations of protein folding allow analysis of the folding process at an unprecedented level of detail. Unfortunately, such simulations have not yet reached their full potential both due to difficulties in sufficiently sampling the microsecond timescales needed for folding, and because the force field used may yield neither the correct dynamical sequence of events nor the folded structure. The ongoing study of protein folding through computational methods thus requires both improvements in the performance of molecular dynamics programs to make longer timescales accessible, and testing of force fields in the context of folding simulations. We report a ten-microsecond simulation of an incipient downhill-folding WW domain mutant along with measurement of a molecular time and activated folding time of 1.5 microseconds and 13.3 microseconds, respectively. The protein simulated in explicit solvent exhibits several metastable states with incorrect topology and does not assume the native state during the present simulations.     

Combining experiment and simulation in protein folding: closing the gap for small model systems

All-atom molecular dynamics (MD) simulations on increasingly powerful computers have been combined with experiments to characterize protein folding in detail over wider time ranges. The folding of small ultrafast folding proteins is being simulated on micros timescales, leading to improved structural predictions and folding rates. To what extent is 'closing the gap' between simulation and experiment for such systems providing insights into general mechanisms of protein folding?     

Methods for molecular dynamics simulations of protein folding/unfolding in solution

All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is compared with experiment. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the experimental evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by experiment occur on timescales accessible with the high-resolution molecular dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temperature quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described.     

Protein folding and unfolding simulations: a new challenge for data mining

One of the unsolved paradigms in molecular biology is the protein folding problem. In recent years, with the identification of several diseases as protein folding disorders and with the explosion of genome information and the need for efficient ways to predict protein structure, protein folding became a central issue in molecular sciences research. Using molecular dynamics unfolding simulations of an amyloidogenic protein--transthyretin--as an example, we put forward a series of ideas on how simulations of this type may be used to infer rules and unfolding behavior in amyloidogenic proteins, and to extrapolate rules for protein folding in different structural classes of proteins. These, in turn, could help in the development of protein structure prediction methods. The need to analyse different proteins and to run multiple simulations creates a huge amount of data which has to be stored, managed, analyzed and shared (database and Grid technology; data mining). Once the data is captured, the next challenge is to find meaningful patterns (associations, correlations, clusters, rules, relationships) among molecular properties, or their relative importance at different stages of the folding or unfolding processes. This clearly puts new and interesting challenges to the bioinformatics community.     

Protein dynamics: From the native to the unfolded state and back again

Simulations to study protein unfolding and folding were performed. The unfolding simulations make use of molecular dynamics and treat an atomic model of barnase in aqueous solvent. The cooperative nature of the unfolding transition and the important role of water are described. The folding simulations are based on a bead model of the protein on a cubic lattice. It is shown for the 27-mer model that a large energy gap between the lowest energy (native) state and the excited states is a necessary and sufficient condition for fast folding.     

Changes of protein stiffness during folding detect protein folding intermediates

Single-molecule force-quench atomic force microscopy (FQ-AFM) is used to detect folding intermediates of a simple protein by detecting changes of molecular stiffness of the protein during its folding process. Those stiffness changes are obtained from shape and peaks of an autocorrelation of fluctuations in end-to-end length of the folding molecule. The results are supported by predictions of the equipartition theorem and agree with existing Langevin dynamics simulations of a simplified model of a protein folding. In the light of the Langevin simulations the experimental data probe an ensemble of random-coiled collapsed states of the protein, which are present both in the force-quench and thermal-quench folding pathways.     

Simulation and experiment at high temperatures: ultrafast folding of a thermophilic protein by nucleation-condensation

We used Phi-value analysis to characterise the transition state for folding of a thermophilic protein at the relatively high temperature of 325 K. PhiF values for the folding of the three-helix bundle, peripheral subunit binding domain from Bacillus stearothermophilus (E3BD) were determined by temperature-jump experiments in the absence of chemical denaturants. E3BD folded in microseconds through a highly diffuse transition state. Excellent agreement was observed between experiment and the results from eight (independent) molecular dynamics simulations of unfolding at 373 K. We used a combination of heteronuclear NMR experiments and molecular dynamics simulations to characterise the denatured ensemble, and found that it contained very little persistent, residual structure. However, those regions that adopt helical structure in the native state were found by simulation to be poised for helix formation in the denatured state. These regions also had significant structure in the transition state for folding. The overall folding pathway appears to be nucleation-condensation.     

Piecewise All-Atom SMD Simulations Reveal Key Secondary Structures in Luciferase Unfolding Pathway

Although the folding of single-domain proteins is well characterized theoretically and experimentally, the folding of large multidomain proteins is less well known. Firefly luciferase, a 550 residue three-domain protein, has been commonly used as a substrate to study chaperone reactions and as a model system for the study of folding of long polypeptide chains, including related phenomena such as cotranslational folding. Despite being characterized by various experimental techniques, the atomic-level contributions of various secondary structures of luciferase to its fold’s mechanical stability remain unknown. Here, we developed a piecewise approach for all-atom steered molecular dynamics simulations to examine specific secondary structures that resist mechanical unfolding while minimizing the amount of computational resources required by the large water box of standard all-atom steered molecular dynamics simulations. We validated the robustness of this approach with a small NI3C protein and used our approach to elucidate the specific secondary structures that provide the largest contributions to luciferase mechanostability. In doing so, we show that piecewise all-atom steered molecular dynamics simulations can provide novel atomic resolution details regarding mechanostability and can serve as a platform for novel mutagenesis studies as well as a point for comparison with high-resolution force spectroscopy experiments.     

Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations

Dimerization of the p53 oligomerization domain involves coupled folding and binding of monomers. To examine the dimerization, we have performed molecular dynamics (MD) simulations of dimer folding from the rate-limiting transition state ensemble (TSE). Among 799 putative transition state structures that were selected from a large ensemble of high-temperature unfolding trajectories, 129 were identified as members of the TSE via calculation of a 50% transmission coefficient from at least 20 room-temperature simulations. This study is the first to examine the refolding of a protein dimer using MD simulations in explicit water, revealing a folding nucleus for dimerization. Our atomistic simulations are consistent with experiment and offer insight that was previously unobtainable.     

Early events in protein folding

Recent advances have significantly increased the time and spectroscopic resolution of protein folding experiments. We can now study the timescale and nature of polypeptide collapse, and how this correlates with secondary and tertiary structure formation. Studies on ultrafast folding proteins and peptides provide experimental benchmarks on a timescale that overlaps directly with that of molecular dynamics simulations. This makes possible direct tests of both simulations and current models of protein folding.     

An independent method for the analysis of protein folding kinetics from all-atom molecular dynamics simulations

We propose a method for extracting useful kinetic information from all-atom molecular dynamics simulations of protein folding. By calculating the time correlation functions between the evolution of different structural properties during the course of the simulation we can determine the endpoint of the reaction and the mechanism by which it occurs. As a test of our method we use thermal denaturation simulations on a 76 residue protein, ubiquitin. The method we present should be used in combination with current techniques for analyzing molecular dynamics trajectories.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.