Assessing and refining molecular dynamics simulations of proteins with nuclear magnetic resonance data

The sophistication of the force fields, algorithms and hardware used for molecular dynamics (MD) simulations of proteins is continuously increasing. No matter how advanced the methodology, however, it is essential to evaluate the appropriateness of the structures sampled in a simulation by comparison with quantitative experimental data. Solution nuclear magnetic resonance (NMR) data are particularly useful for checking the quality of protein simulations, as they provide both structural and dynamic information on a variety of temporal and spatial scales. Here, various features and implications of using NMR data to validate and bias MD simulations are outlined, including an overview of the different types of NMR data that report directly on structural properties and of relevant simulation techniques. The focus throughout is on how to properly account for conformational averaging, particularly within the context of the assumptions inherent in the relationships that link NMR data to structural properties.     

Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by F?rster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.     

Contribution of Fluorophore Dynamics and Solvation to Resonant Energy Transfer in Protein-DNA Complexes: A Molecular-Dynamics Study

In Förster resonance energy transfer (FRET) experiments, extracting accurate structural information about macromolecules depends on knowing the positions and orientations of donor and acceptor fluorophores. Several approaches have been employed to reduce uncertainties in quantitative FRET distance measurements. Fluorophore-position distributions can be estimated by surface accessibility (SA) calculations, which compute the region of space explored by the fluorophore within a static macromolecular structure. However, SA models generally do not take fluorophore shape, dye transition-moment orientation, or dye-specific chemical interactions into account. We present a detailed molecular-dynamics (MD) treatment of fluorophore dynamics for an ATTO donor/acceptor dye pair and specifically consider as case studies dye-labeled protein-DNA intermediates in Cre site-specific recombination. We carried out MD simulations in both an aqueous solution and glycerol/water mixtures to assess the effects of experimental solvent systems on dye dynamics. Our results unequivocally show that MD simulations capture solvent effects and dye-dye interactions that can dramatically affect energy transfer efficiency. We also show that results from SA models and MD simulations strongly diverge in cases where donor and acceptor fluorophores are in close proximity. Although atomistic simulations are computationally more expensive than SA models, explicit MD studies are likely to give more realistic results in both homogeneous and mixed solvents. Our study underscores the model-dependent nature of FRET analyses, but also provides a starting point to develop more realistic in silico approaches for obtaining experimental ensemble and single-molecule FRET data.     

Contribution of Fluorophore Dynamics and Solvation to Resonant Energy Transfer in Protein-DNA Complexes: A Molecular-Dynamics Study

In Förster resonance energy transfer (FRET) experiments, extracting accurate structural information about macromolecules depends on knowing the positions and orientations of donor and acceptor fluorophores. Several approaches have been employed to reduce uncertainties in quantitative FRET distance measurements. Fluorophore-position distributions can be estimated by surface accessibility (SA) calculations, which compute the region of space explored by the fluorophore within a static macromolecular structure. However, SA models generally do not take fluorophore shape, dye transition-moment orientation, or dye-specific chemical interactions into account. We present a detailed molecular-dynamics (MD) treatment of fluorophore dynamics for an ATTO donor/acceptor dye pair and specifically consider as case studies dye-labeled protein-DNA intermediates in Cre site-specific recombination. We carried out MD simulations in both an aqueous solution and glycerol/water mixtures to assess the effects of experimental solvent systems on dye dynamics. Our results unequivocally show that MD simulations capture solvent effects and dye-dye interactions that can dramatically affect energy transfer efficiency. We also show that results from SA models and MD simulations strongly diverge in cases where donor and acceptor fluorophores are in close proximity. Although atomistic simulations are computationally more expensive than SA models, explicit MD studies are likely to give more realistic results in both homogeneous and mixed solvents. Our study underscores the model-dependent nature of FRET analyses, but also provides a starting point to develop more realistic in silico approaches for obtaining experimental ensemble and single-molecule FRET data.     

A statistical approach to the interpretation of molecular dynamics simulations of calmodulin equilibrium dynamics

A sample of 35 independent molecular dynamics (MD) simulations of calmodulin (CaM) equilibrium dynamics was prepared from different but equally plausible initial conditions (20 simulations of the wild-type protein and 15 simulations of the D129N mutant). CaM's radius of gyration and backbone mean-square fluctuations were analyzed for the effect of the D129N mutation, and simulations were compared with experiments. Statistical tests were employed for quantitative comparisons at the desired error level. The computational model predicted statistically significant compaction of CaM relative to the crystal structure, consistent with the results of small-angle X-ray scattering (SAXS) experiments. This effect was not observed in several previously reported studies of (Ca2+)(4)-CaM, which relied on a single MD run. In contrast to radius of gyration, backbone mean-square fluctuations showed a distinctly non-normal and positively skewed distribution for nearly all residues. Furthermore, the D129N mutation affected the backbone dynamics in a complex manner and reduced the mobility of Glu123, Met124, Ile125, Arg126, and Glu127 located in the adjacent alpha-helix G. The implications of these observations for the comparisons of MD simulations with experiments are discussed. The proposed approach may be useful in studies of protein equilibrium dynamics where MD simulations fall short of properly sampling the conformational space, and when the comparison with experiments is affected by the reproducibility of the computational model.     

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from ¹⁵N relaxation and side-chain dynamics from ¹³C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.     

A short guide for molecular dynamics simulations of RNA systems

As a result of important methodological advances and of the rapid growth of experimental data, the number of molecular dynamics (MD) simulations related to RNA systems has significantly increased. However, such MD simulations are not straightforward and great care has to be exerted during the setup stage in order to choose the appropriate MD package, force fields and ionic conditions. Furthermore, the choice and a correct evaluation of the main characteristics of the starting structure are primordial for the generation of informative and reliable MD trajectories since experimental structures are not void of inaccuracies and errors. The aim of this review is to provide, through numerous examples, practical guidelines for the setup of MD simulations, the choice of ionic conditions and the detection and correction of experimental inaccuracies in order to start MD simulations of nucleic acid systems under the best auspices.     

Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation

The ubiquitin associated domain of p62 is a small three-helix bundle of approximately 50 residues that mediates the recognition of polyubiquitin chains and ubiquitylated substrates. The solution structure of a 52 residue construct containing this domain has been characterized using heteronuclear nuclear magnetic resonance (NMR) methods. The resulting ensemble of NMR-derived structures was used in molecular dynamics (MD) simulations to investigate the equilibrium conformation and dynamics of this domain. NOE and (15)N relaxation data have been used to validate the structural ensemble produced by the MD simulations and show a good correlation for residues in regions of secondary structure. A similar approach was taken using an ensemble of structures from the MD simulations to calculate electronic circular dichroism (CD) and IR spectra from first principles with an encouraging correlation with the experimental CD and IR data.     

Interpretation of H-NMR Experiments on the Orientation of the Transmembrane Helix WALP23 by Computer Simulations

Orientation, dynamics, and packing of transmembrane helical peptides are important determinants of membrane protein structure, dynamics, and function. Because it is difficult to investigate these aspects by studying real membrane proteins, model transmembrane helical peptides are widely used. NMR experiments provide information on both orientation and dynamics of peptides, but they require that motional models be interpreted. Different motional models yield different interpretations of quadrupolar splittings (QS) in terms of helix orientation and dynamics. Here, we use coarse-grained (CG) molecular dynamics (MD) simulations to investigate the behavior of a well-known model transmembrane peptide, WALP23, under different hydrophobic matching/mismatching conditions. We compare experimental ²H-NMR QS (directly measured in experiments), as well as helix tilt angle and azimuthal rotation (not directly measured), with CG MD simulation results. For QS, the agreement is significantly better than previously obtained with atomistic simulations, indicating that equilibrium sampling is more important than atomistic details for reproducing experimental QS. Calculations of helix orientation confirm that the interpretation of QS depends on the motional model used. Our simulations suggest that WALP23 can form dimers, which are more stable in an antiparallel arrangement. The origin of the preference for the antiparallel orientation lies not only in electrostatic interactions but also in better surface complementarity. In most cases, a mixture of monomers and antiparallel dimers provides better agreement with NMR data compared to the monomer and the parallel dimer. CG MD simulations allow predictions of helix orientation and dynamics and interpretation of QS data without requiring any assumption about the motional model.     

Molecular dynamics simulations of RNA: an in silico single molecule approach

RNA molecules are now known to be involved in the processing of genetic information at all levels, taking on a wide variety of central roles in the cell. Understanding how RNA molecules carry out their biological functions will require an understanding of structure and dynamics at the atomistic level, which can be significantly improved by combining computational simulation with experiment. This review provides a critical survey of the state of molecular dynamics (MD) simulations of RNA, including a discussion of important current limitations of the technique and examples of its successful application. Several types of simulations are discussed in detail, including those of structured RNA molecules and their interactions with the surrounding solvent and ions, catalytic RNAs, and RNA-small molecule and RNA-protein complexes. Increased cooperation between theorists and experimentalists will allow expanded judicious use of MD simulations to complement conceptually related single molecule experiments. Such cooperation will open the door to a fundamental understanding of the structure-function relationships in diverse and complex RNA molecules. .     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Molecular dynamics simulation of hen egg white lysozyme: a test of the GROMOS96 force field against nuclear magnetic resonance data

Biomolecular force fields for use in molecular dynamics (MD) simulations of proteins, DNA, or membranes are generally parametrized against ab initio quantum-chemical and experimental data for small molecules. The application of a force field in a simulation of a biomolecular system, such as a protein in solution, may then serve as a test of the quality and transferability of the force field. Here, we compare various properties obtained from two MD simulations of the protein hen egg white lysozyme (HEWL) in aqueous solution using the latest version, GROMOS96, of the GROMOS force field and an earlier version, GROMOS87+, with data derived from nuclear magnetic resonance (NMR) experiments: NOE atom-atom distance bounds, (3)J(HNalpha)-coupling constants, and backbone and side-chain order parameters. The convergence of these quantities over a 2-ns period is considered, and converged values are compared to experimental ones. The GROMOS96 simulation shows better agreement with the NMR data and also with the X-ray crystal structure of HEWL than the GROMOS87+ simulation, which was based on an earlier version of the GROMOS force field.     

Water and ion binding around RNA and DNA (C,G) oligomers

The dynamics, hydration, and ion-binding features of two duplexes, the A(r(CG)(12)) and the B(d(CG)(12)), in a neutralizing aqueous environment with 0.25 M added KCl have been investigated by molecular dynamics (MD) simulations. The regular repeats of the same C=G base-pair motif have been exploited as a statistical alternative to long MD simulations in order to extend the sampling of the conformational space. The trajectories demonstrate the larger flexibility of DNA compared to RNA helices. This flexibility results in less well defined hydration patterns around the DNA than around the RNA backbone atoms. Yet, 22 hydration sites are clearly characterized around both nucleic acid structures. With additional results from MD simulations, the following hydration scale for C=G pairs can be deduced: A-DNA相似文献   

Molecular dynamics simulations of Guanine quadruplex loops: advances and force field limitations

A computational analysis of d(GGGGTTTTGGGG)(2) guanine quadruplexes containing either lateral or diagonal four-thymidine loops was carried out using molecular dynamics (MD) simulations in explicit solvent, locally enhanced sampling (LES) simulations, systematic conformational search, and free energy molecular-mechanics, Poisson Boltzmann, surface area (MM-PBSA) calculations with explicit inclusion of structural monovalent cations. The study provides, within the approximations of the applied all-atom additive force field, a qualitatively complete analysis of the available loop conformational space. The results are independent of the starting structures. Major conformational transitions not seen in conventional MD simulations are observed when LES is applied. The favored LES structures consistently provide lower free energies (as estimated by molecular-mechanics, Poisson Boltzmann, surface area) than other structures. Unfortunately, the predicted optimal structure for the diagonal loop arrangement differs substantially from the atomic resolution experiments. This result is attributed to force field deficiencies, such as the potential misbalance between solute-cation and solvent-cation terms. The MD simulations are unable to maintain the stable coordination of the monovalent cations inside the diagonal loops as reported in a recent x-ray study. The optimal diagonal and lateral loop arrangements appear to be close in energy although a proper inclusion of the loop monovalent cations could stabilize the diagonal architecture.     

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC(50) = 0.28 microM), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available.