Predicting molecular properties of α-synuclein using force fields for intrinsically disordered proteins

Independent force field validation is an essential practice to keep track of developments and for performing meaningful Molecular Dynamics simulations. In this work, atomistic force fields for intrinsically disordered proteins (IDP) are tested by simulating the archetypical IDP α-synuclein in solution for 2.5 μs. Four combinations of protein and water force fields were tested: ff19SB/OPC , ff19SB/TIP4P-D , ff03CMAP/TIP4P-D , and a99SB-disp/TIP4P-disp , with four independent repeat simulations for each combination. We compare our simulations to the results of a 73 μs simulation using the a99SB-disp/TIP4P-disp combination, provided by D. E. Shaw Research. From the trajectories, we predict a range of experimental observations of α-synuclein and compare them to literature data. This includes protein radius of gyration and hydration, intramolecular distances, NMR chemical shifts, and ³J-couplings. Both ff19SB/TIP4P-D and a99SB-disp/TIP4P-disp produce extended conformational ensembles of α-synuclein that agree well with experimental radius of gyration and intramolecular distances while a99SB-disp/TIP4P-disp reproduces a balanced α-synuclein secondary structure content. It was found that ff19SB/OPC and ff03CMAP/TIP4P-D produce overly compact conformational ensembles and show discrepancies in the secondary structure content compared to the experimental data.     

Examination of the quality of various force fields and solvation models for the equilibrium simulations of GA88 and GB88

Elucidating the relationship between sequence and conformation is essential for the understanding of functions of proteins. While sharing 88 % sequence identity and differing by only seven residues, GA88 and GB88 have completely different structures and serve as ideal systems for investigating the relationship between sequence and function. Benefiting from the continuous advancement of the computational ability of modern computers, molecular dynamics (MD) simulation is now playing an increasingly important role in the study of proteins. However, the reliability of MD simulations is limited by the accuracy of the force fields and solvent model approximations. In this work, several AMBER force fields (AMBER03, AMBER99SB, AMBER12SB, AMBER14SB, AMBER96) and solvent models (TIP3P, IGB5, IGB7, IGB8) have been employed in the simulations of GA88 and GB88. The statistical results from 19 simulations show that GA88 and GB88 both adopt more compact structures than the native structures. GB88 is more stable than GA88 regardless of the force fields and solvent models utilized. Most of the simulations overestimated the salt bridge interaction. The combination of AMBER14SB force field and IGB8 solvent model shows the best overall performance in the simulations of both GA88 and GB88. AMBER03 and AMBER12SB also yield reasonable results but only in the TIP3P explicit solvent model.     

Improved side‐chain torsion potentials for the Amber ff99SB protein force field

Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side‐chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha‐helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side‐chain torsion potentials of these residues to match new, high‐level quantum‐mechanical calculations. Finally, we used microsecond‐timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side‐chain conformations. The new force field, which we have termed Amber ff99SB‐ILDN, exhibits considerably better agreement with the NMR data. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Toward a predictive understanding of slow methyl group dynamics in proteins

The development of the most recent generation of molecular mechanics force fields promises an increasingly predictive understanding of the protein dynamics-function relationship. Based on extensive validation against various types of experimental data, the AMBER force field ff99SB was benchmarked in recent years as a favorable force field for protein simulations. Recent improvements of the side chain and backbone potentials, made by different groups, led to the ff99SB-ILDN and ff99SBnmr1 force fields, respectively. The combination of these potentials into a unified force field, termed ff99SBnmr1-ILDN, was used in this study to perform a microsecond time scale molecular dynamics simulation of free ubiquitin in explicit solvent for validation against an extensive set of experimental NMR methyl group residual dipolar couplings. Our results show a high level of consistency between the experimental data and the values predicted from the molecular dynamics trajectory reflecting a systematically improved performance of ff99SBnmr1-ILDN over the original ff99SB force field. Moreover, the unconstrained ff99SBnmr1-ILDN MD ensemble achieves a similar level of agreement as the recently introduced EROS ensemble, which was constructed based on a large body of NMR data as constraints, including the methyl residual dipolar couplings. This suggests that ff99SBnmr1-ILDN provides a high-quality representation of the motions of methyl-bearing protein side chains, which are sensitive probes of protein-protein and protein-ligand interactions.     

Molecular dynamics guided study of salt bridge length dependence in both fluorinated and non-fluorinated parallel dimeric coiled-coils

The alpha-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of the coiled-coil motifs, including those modified by fluorination, several fluorinated and nonfluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiled-coil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER used a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, and ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly, better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller L-ornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled-coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine when compared with 5,5,5,5',5',5'-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5',5',5'-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may provide a driving force for coiled-coil dimer formation. Estimation of the ellipticity at 222 nm from a series of snapshots from the MD simulations with DicroCalc shows distinct increases in the ellipticity when the coiled-coil is fluorinated, which suggests that the helicity in the folded coiled-coils is greater when fluorinated.     

Force field influences in beta-hairpin folding simulations

All-atom force fields are now routinely used for more detailed understanding of protein folding mechanisms. However, it has been pointed out that use of all-atom force fields does not guarantee more accurate representations of proteins; in fact, sometimes it even leads to biased structural distributions. Indeed, several issues remain to be solved in force field developments, such as accurate treatment of implicit solvation for efficient conformational sampling and proper treatment of backbone interactions for secondary structure propensities. In this study, we first investigate the quality of several recently improved backbone interaction schemes in AMBER for folding simulations of a beta-hairpin peptide, and further study their influences on the peptide's folding mechanism. Due to the significant number of simulations needed for a thorough analysis of tested force fields, the implicit Poisson-Boltzmann solvent was used in all simulations. The chosen implicit solvent was found to be reasonable for studies of secondary structures based on a set of simulations of both alpha-helical and beta-hairpin peptides with the TIP3P explicit solvent as benchmark. Replica exchange molecular dynamics was also utilized for further efficient conformational sampling. Among the tested AMBER force fields, ff03 and a revised ff99 force field were found to produce structural and thermodynamic data in comparably good agreement with the experiment. However, detailed folding pathways, such as the order of backbone hydrogen bond zipping and the existence of intermediate states, are different between the two force fields, leading to force field-dependent folding mechanisms.     

Computational studies on carbohydrates: solvation studies on maltose and cyclomaltooligosaccharides (cyclodextrins) using a DFT/ab initio-derived empirical force field, AMB99C

An empirical force field, denoted AMB99C, has been used to study molecular properties of alpha-(1-->4)-linked carbohydrates in solution. AMB99C was parameterized using structural and energetic parameters from density functional ab initio methodology. In this work we examine the solution behavior of the beta anomer of maltose and cyclohexa-, cyclohepta-, and cyclooctaamyloses (alpha-, beta-, and gamma-cyclodextrins or alpha-, beta-, and gamma-CDs, respectively), as well as of two larger (DP 10, epsilon-CD; DP 21) cyclomaltooligosaccharides, CA10 and CA21. Experimental data used for comparison purposes include X-ray structures, small-angle scattering radius of gyration values, NMR nuclear Overhauser enhancements (NOEs), and proton coupling constants. Molecular dynamics simulations were carried out using explicit water molecules (TIP3P) to establish equilibrium populations of conformations in solution, and these results are compared with other calculated values and a variety of experimental parameters, such as average H-1-H-4' distances between the rings in beta-maltose, and the primary hydroxyl groups' conformational populations. Medium-to-large cyclomaltooligosaccharide molecules were studied to test for glucose ring puckering and stability of kinked and 'flipped' conformations. The results of the solvation studies are in excellent agreement with experimental structural parameters.     

Evaluating Molecular Mechanical Potentials for Helical Peptides and Proteins

Multiple variants of the AMBER all-atom force field were quantitatively evaluated with respect to their ability to accurately characterize helix-coil equilibria in explicit solvent simulations. Using a global distributed computing network, absolute conformational convergence was achieved for large ensembles of the capped A₂₁ and F_s helical peptides. Further assessment of these AMBER variants was conducted via simulations of a flexible 164-residue five-helix-bundle protein, apolipophorin-III, on the 100 ns timescale. Of the contemporary potentials that had not been assessed previously, the AMBER-99SB force field showed significant helix-destabilizing tendencies, with beta bridge formation occurring in helical peptides, and unfolding of apolipophorin-III occurring on the tens of nanoseconds timescale. The AMBER-03 force field, while showing adequate helical propensities for both peptides and stabilizing apolipophorin-III, (i) predicts an unexpected decrease in helicity with ALA→ARG⁺ substitution, (ii) lacks experimentally observed 3₁₀ helical content, and (iii) deviates strongly from average apolipophorin-III NMR structural properties. As is observed for AMBER-99SB, AMBER-03 significantly overweighs the contribution of extended and polyproline backbone configurations to the conformational equilibrium. In contrast, the AMBER-99φ force field, which was previously shown to best reproduce experimental measurements of the helix-coil transition in model helical peptides, adequately stabilizes apolipophorin-III and yields both an average gyration radius and polar solvent exposed surface area that are in excellent agreement with the NMR ensemble.     

Microsecond simulations of mdm2 and its complex with p53 yield insight into force field accuracy and conformational dynamics

The p53‐MDM2 complex is both a major target for cancer drug development and a valuable model system for computational predictions of protein‐ligand binding. To investigate the accuracy of molecular simulations of MDM2 and its complex with p53, we performed a number of long (200 ns to 1 µs) explicit‐solvent simulations using a range of force fields. We systematically compared nine popular force fields (AMBER ff03, ff12sb, ff14sb, ff99sb, ff99sb‐ildn, ff99sb‐ildn‐nmr, ff99sb‐ildn‐phi, CHARMM22*, and CHARMM36) against experimental chemical shift data, and found similarly accurate results, with microsecond simulations achieving better agreement compared to 200‐ns trajectories. Although the experimentally determined apo structure has a closed binding cleft, simulations in all force fields suggest the apo state of MDM2 is highly flexible, and able to sample holo‐like conformations, consistent with a conformational selection model. Initial structuring of the MDM2 lid region, known to competitively bind the binding cleft, is also observed in long simulations. Taken together, these results show molecular simulations can accurately sample conformations relevant for ligand binding. We expect this study to inform future computational work on folding and binding of MDM2 ligands. Proteins 2015; 83:1665–1676. © 2015 Wiley Periodicals, Inc.     

Evaluating amber force fields using computed NMR chemical shifts

NMR chemical shifts can be computed from molecular dynamics (MD) simulations using a template matching approach and a library of conformers containing chemical shifts generated from ab initio quantum calculations. This approach has potential utility for evaluating the force fields that underlie these simulations. Imperfections in force fields generate flawed atomic coordinates. Chemical shifts obtained from flawed coordinates have errors that can be traced back to these imperfections. We use this approach to evaluate a series of AMBER force fields that have been refined over the course of two decades (ff94, ff96, ff99SB, ff14SB, ff14ipq, and ff15ipq). For each force field a series of MD simulations are carried out for eight model proteins. The calculated chemical shifts for the ¹H, ¹⁵N, and ¹³C^a atoms are compared with experimental values. Initial evaluations are based on root mean squared (RMS) errors at the protein level. These results are further refined based on secondary structure and the types of atoms involved in nonbonded interactions. The best chemical shift for identifying force field differences is the shift associated with peptide protons. Examination of the model proteins on a residue by residue basis reveals that force field performance is highly dependent on residue position. Examination of the time course of nonbonded interactions at these sites provides explanations for chemical shift differences at the atomic coordinate level. Results show that the newer ff14ipq and ff15ipq force fields developed with the implicitly polarized charge method perform better than the older force fields.     

Are Current Molecular Dynamics Force Fields too Helical?

Accurate force fields are essential for the success of molecular dynamics simulations. In apparent contrast to the conformational preferences of most force fields, recent NMR experiments suggest that short polyalanine peptides in water populate the polyproline II structure almost exclusively. To investigate this apparent contradiction, with its ramifications for the assessment of molecular force fields and the structure of unfolded proteins, we performed extensive simulations of Ala₅ in water (∼5 μs total time), using twelve different force fields and three different peptide terminal groups. Using either empirical or density-functional-based Karplus relations for the J-couplings, we find that most current force fields do overpopulate the α-region, with quantitative results depending on the choice of Karplus relation and on the peptide termini. Even after reweighting to match experiment, we find that Ala₅ retains significant α- and β-populations. In fact, several force fields match the experimental data well before reweighting and have a significant helical population. We conclude that radical changes to the best current force fields are not necessary, based on the NMR data. Nevertheless, experiments on short peptides open the way toward the systematic improvement of current simulation models.     

Can current force fields reproduce ring puckering in 2-O-sulfo-α-l-iduronic acid? A molecular dynamics simulation study

The monosaccharide 2-O-sulfo-α-l-iduronic acid (IdoA2S) is one of the major components of glycosaminoglycans. The ability of molecular mechanics force fields to reproduce ring-puckering conformational equilibrium is important for the successful prediction of the free energies of interaction of these carbohydrates with proteins. Here we report unconstrained molecular dynamics simulations of IdoA2S monosaccharide that were carried out to investigate the ability of commonly used force fields to reproduce its ring conformational flexibility in aqueous solution. In particular, the distribution of ring conformer populations of IdoA2S was determined. The GROMOS96 force field with the SPC/E water potential can predict successfully the dominant skew-boat to chair conformational transition of the IdoA2S monosaccharide in aqueous solution. On the other hand, the GLYCAM06 force field with the TIP3P water potential sampled transitional conformations between the boat and chair forms. Simulations using the GROMOS96 force field showed no pseudorotational equilibrium fluctuations and hence no inter-conversion between the boat and twist boat ring conformers. Calculations of theoretical proton NMR coupling constants showed that the GROMOS96 force field can predict the skew-boat to chair conformational ratio in good agreement with the experiment, whereas GLYCAM06 shows worse agreement. The omega rotamer distribution about the C5-C6 bond was predicted by both force fields to have torsions around 10°, 190°, and 360°.     

Validation of molecular force field parameters for peptides including isomerized amino acids

Recently, stereoinversions and isomerizations of amino acid residues in the proteins of living beings have been observed. Because isomerized amino acids cause structural changes and denaturation of proteins, isomerizations of amino acid residues are suspected to cause age‐related diseases. In this study, AMBER molecular force field parameters were tested by using computationally generated nonapeptides and tripeptides including stereoinverted and/or isomerized amino acid residues. Energy calculations by using density functional theory were also performed for comparison. Although the force field parameters were developed by parameter fitting for l ‐α‐amino acids, the accuracy of the computational results for d ‐amino acids and β‐amino acids was comparable to those for l ‐α‐amino acids. The conformational energies for tripeptides calculated by using density functional theory were reproduced more accurately than those for nonapeptides calculated by using the molecular mechanical force field. The evaluations were performed for the ff99SB, ff03, ff12SB, and the latest ff14SB force field parameters.     

Derivation of original RESP atomic partial charges for MD simulations of the LDAO surfactant with AMBER: applications to a model of micelle and a fragment of the lipid kinase PI4KA

In this paper, we describe the derivation and the validation of original RESP atomic partial charges for the N, N-dimethyl-dodecylamine oxide (LDAO) surfactant. These charges, designed to be fully compatible with all the AMBER force fields, are at first tested against molecular dynamics simulations of pure LDAO micelles and with a fragment of the lipid kinase PIK4A (DI) modeled with the QUARK molecular modeling server. To model the micelle, we used two distinct AMBER force fields (i.e. Amber99SB and Lipid14) and a variety of starting conditions. We find that the micelle structural properties (such as the shape, size, the LDAO headgroup hydration, and alkyl chain conformation) slightly depend on the force field but not on the starting conditions and more importantly are in good agreement with experiments and previous simulations. We also show that the Lipid14 force field should be used instead of the Amber99SB one to better reproduce the C(sp3)C(sp3)C(sp3)C(sp3) conformation in the surfactant alkyl chain. Concerning the simulations with LDAO-DI protein, we carried out different runs at two NaCl concentrations (i.e. 0 and 300 mM) to mimic, in the latter case, the experimental conditions. We notice a small dependence of the simulation results with the LDAO parameters and the salt concentration. However, we find that in the simulations, three out of four tryptophans of the DI protein are not accessible to water in agreement with our fluorescence spectroscopy experiments reported in the paper.     

The Application of the Reaction-Field Method to the Calculation of Dielectric Constants

Abstract

The behaviour of the popular TIP3P water model has been investigated using both molecular dynamics and Monte Carlo simulation procedures. Long-range electrostatic interactions were included through a reaction-field treatment, and the nonbonded interactions were either truncated at the cutoff distance, or smoothly scaled to zero using a switching function. The thermodynamic observables, and in particular the dipole-dipole correlation functions, are found to differ between the two simulation techniques if a rigid nonbonded cutoff is applied. However, use of a switching function gives exact agreement between the simulation methodologies. This difference is ascribed to the effect of energy pumping in the molecular dynamics simulations, and suggests that dielectric constants calculated using this simulation method with the fluctuation procedure in conjunction with a reaction field should be reappraised. Thus the Monte Carlo simulation procedure offers a number of intrinsic advantages over molecular dynamics for the calculation of dielectric constants with a reaction field. The most precise value for the dielectric constant of TIP3P is calculated to be 102 ± 3 at 298 K.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

Comparison of force fields for Alzheimer's A : A case study for intrinsically disordered proteins

Intrinsically disordered proteins are essential for biological processes such as cell signalling, but are also associated to devastating diseases including Alzheimer's disease, Parkinson's disease or type II diabetes. Because of their lack of a stable three‐dimensional structure, molecular dynamics simulations are often used to obtain atomistic details that cannot be observed experimentally. The applicability of molecular dynamics simulations depends on the accuracy of the force field chosen to represent the underlying free energy surface of the system. Here, we use replica exchange molecular dynamics simulations to test five modern force fields, OPLS, AMBER99SB, AMBER99SB*ILDN, AMBER99SBILDN‐NMR and CHARMM22*, in their ability to model Aβ₄₂, an intrinsically disordered peptide associated with Alzheimer's disease, and compare our results to nuclear magnetic resonance (NMR) experimental data. We observe that all force fields except AMBER99SBILDN‐NMR successfully reproduce local NMR observables, with CHARMM22* being slightly better than the other force fields.