All-atom molecular dynamics study of EAK16 peptide: the effect of pH on single-chain conformation,dimerization and self-assembly behavior

Single-chain equilibrium conformation and dimerization of the three types of ionic EAK16 peptide are studied under three pH conditions using all-atom molecular dynamics simulations. It is found that both the single-chain conformation and the dimerization process of EAK16-IV are considerably different from those of the two other types, EAK16-I and EAK16-II. The value of pH is found to have a stronger effect on the single-chain conformation and dimerization of EAK16-IV. It is shown that in addition to the charge pattern on the peptide chains, the size of the side chains of the charged amino acids plays role in the conformation of the peptide chains and their dimerization. The results shed light on the pH-dependent self-assembly behavior of EAK16 peptide in the bulk solution, which has been reported in the literature.     

Self-assembly of the ionic peptide EAK16: the effect of charge distributions on self-assembly

Amphiphilic peptides suspended in aqueous solution display a rich set of aggregation behavior. Molecular-level studies of relatively simple amphiphilic molecules under controlled conditions are an essential step toward a better understanding of self-assembly phenomena of naturally occurring peptides/proteins. Here, we study the influence of molecular architecture and interactions on the self-assembly of model peptides (EAK16s), using both experimental and theoretical approaches. Three different types of EAK16 were studied: EAK16-I, -II, and -IV, which have the same amino acid composition but different amino acid sequences. Atomic force microscopy confirms that EAK16-I and -II form fibrillar assemblies, whereas EAK16-IV forms globular structures. The Fourier transform infrared spectrum of EAK16-IV indicates the possible formation of a beta-turn structure, which is not found in EAK16-I and -II. Our theoretical and numerical studies suggest the underlying mechanism behind these observations. We show that the hairpin structure is energetically stable for EAK16-IV, whereas the chain entropy of EAK16-I and -II favors relatively stretched conformations. Our combined experimental and theoretical approaches provide a clear picture of the interplay between single-chain properties, as determined by peptide sequences (or charge distributions), and the emerging structure at the nano (or more coarse-grained) level.     

Insights into aggregation dynamics of NACore peptides from coarse-grained simulations

Alpha(α)-synuclein is closely related to the pathogenesis of Parkinson's disease (PD). The NACore, a fragment of α-synuclein, is considered to be the key region of α-synuclein that causes PD. The aggregation dynamics of NACores are studied via coarse-grained molecular dynamics simulations. We find that NACores can self-assemble into a large cluster at high concentrations. The aggregation dynamics can be divided into three stages. The growth kinetics for the first and second stages follows the power law, S_max ~ t^γ, with the second stage faster than the first one. The characteristic lifetime for the high concentration is 40 times larger than that for the low concentration, implying the low fluidity. Understanding the aggregation dynamics of NACores is helpful to develop drugs for therapeutic prevention and intervention.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

Transitions from alpha to pi helix observed in molecular dynamics simulations of synthetic peptides

Molecular dynamics simulations were carried out for three 13-residue peptides of the form AcNH-A-A-E-X-A-E-A-H-A-A-E-K-A-CONH(2) with X = A, F, and W. All three peptides exhibited unexpected dynamical behavior, undergoing a transition from an alpha-helical to a pi-helical structure in the course of 5-ns trajectories in aqueous solution. Analysis of peptide length, accessible surface, interaction energies, hydrogen bonding, and dihedral angles was consistent with alpha --> pi transitions at 2800, 500, and 800 ps for X = A, F and W, respectively. The transitions occurred sequentially and cooperatively, propagating from the C- to the N-terminus for X = A and W and from the center toward both termini for X = F. The time scale of the overall transition ranged from 300 to 500 ps. For all three peptides the backbone structural transition was accompanied by a concerted rearrangement of the charged side chains, including a 3 A increase in the distance between carboxylate groups of Glu-3 and Glu-6. During the transition the peptide backbone hydrogen-bonding patterns were disrupted at the interface between the alpha-helical and nascent pi-helical regions, with peptide groups forming water-bridged hydrogen bonds. The peptide structures exhibited significant fluidity, with individual residues sampling alpha-, pi-, and 3(10)-helical conformations, as well as a "coil" state, without any intramolecular hydrogen bonds. The studied peptides have been designed to form alpha-helices when incorporated in novel hemoprotein model compounds, peptide-sandwiched mesohemes, which consist of two identical peptides covalently attached to an Fe(III) mesoporphyrin [Liu, D., Williamson, D. A., Kennedy, M. L., Williams, T. D., Morton, M. M., and Benson, D. R. (1999) J. Am. Chem. Soc. 121, 11798-11812]. The possibility of adopting pi-helical structures by the constituent peptides may influence the properties of the hemoprotein models.     

New insights into the structure of abasic DNA from molecular dynamics simulations

Abasic (AP) sites constitute a common form of DNA damage, arising from the spontaneous or enzymatic breakage of the N-glycosyl bond and the loss of a nucleotide base. To examine the effects of such damage on DNA structure, especially in the vicinity of the abasic sugar, four 1.5 ns molecular dynamics simulations of double-helical DNA dodecamers with and without a single abasic (tetrahydrofuran, X) lesion in a 5′-d(CXT) context have been performed and analyzed. The results indicate that the abasic site does not maintain a hole or gap in the DNA, but instead perturbs the canonical structure and induces additional flexibility close to the abasic site. In the apurinic simulations (i.e., when a pyrimidine is opposite the AP site), the abasic sugar flipped in and out of the minor groove, and the gap was water filled, except during the occurrence of a novel non-Watson–Crick C-T base pair across the abasic site. The apyrimidinic gap was not penetrated by water until the abasic sugar flipped out and remained extrahelical. Both AP helices showed kinks of 20–30° at the abasic site. The Watson–Crick hydrogen bonds are more transient throughout the DNA double helices containing an abasic site. The abasic sugar displayed an unusually broad range of sugar puckers centered around the northern pucker. The increased motion of the bases and backbone near the abasic site appear to correlate with sequence-dependent helical stability. The data indicate that abasic DNA contorts more easily and in specific ways relative to unmodified DNA, an aspect likely to be important in abasic site recognition and hydrolysis.     

Characterization of molecular structures and properties of polyurethanes using molecular dynamics simulations

Thermotropic polyurethanes with mesogenic groups in side chains were prepared from two diisocyanates and four diols with stoichiometric ratios of reactive isocyanate (NCO) and hydroxy (OH) groups. Their thermal behavior was determined by differential scanning calorimetry. The effect of structure modifications of the diisocyanates and diols, in particular changes in the mesogen, were investigated. Introduction of mesogenic segments into the polymers suppresses the ordering. Stiff end substituents (phenyl and alkoxy groups) of the mesogens stabilize the mesophases to such an extent that the negative influence of long polymer chains is compensated and the liquid-crystalline properties are recovered. All-atom molecular dynamics simulations in the Cerius² modeling environment were carried out to characterize the structures of the polymers. Analysis of the dynamic trajectories at 20, 100, 120 and 170 °C revealed changes in conformation of macromolecules, which correlate with DSC measurements.Figure Example of structure relaxation of D4/TDI molecule at indicated simulation times (temperature 20 °C): a complete structure; b backbone structure; c top view of molecule     

Improved molecular dynamics simulations for the determination of peptide structures

In this article a few methods or modifications proven to be useful in the conformational examination of peptides and related molecules by molecular dynamics are illustrated. The first is the explicit use of organic solvents in the simulations. For many cases such solvents are appropriate since the nmr measurements (or other experimental observations) were carried out in the same solvent. Here, the use of dimethylsulfoxide and chloroform in molecular dynamics is described, with some advantages of the use of these solvents highlighted. A constant allowing for the scaling of the nonbonded interactions of the force field, an idea previously employed in distance geometry and simulated annealing, has been implemented. The usefulness of this method is that when the nonbonded term is turned to zero, atoms can pass through each other, while the connectivity of the molecule is maintained. It will be shown that such simulations, if a sufficient driving force is present (i.e., nuclear Overhauser effects restraints), can produce the correct stereoconfiguration (i.e., chiral center) as well as configurational isomer (i.e., cis/trans isomers). Lastly, a penalty term for coupling constants directly related to the Karplus curve has been plemented into the potential energy force field. The advantages of this method over the commonly used dihedral angle restraining are discussed. In particular, it is shown that with more than one coupling constant about a dihedral angle a great reduction of the allowed conformational space is obtained. © 1993 John Wiley & Sons, Inc.     

Partition of protein solvation into group contributions from molecular dynamics simulations

Linear response theory coupled to molecular dynamics simulations with an explicit solvent representation is used to derive fractional contributions of amino acid residues to the solvation of proteins. The new fractional methods developed here are compared with standard approaches based on empirical 1D and 3D statistical potentials, as well as with estimates obtained from the analysis of classical molecular interaction potentials. The new fractional methods, which have a clear physical basis and explicitly account for the effects due to protein structure and flexibility, provide an accurate picture of the contribution to solvation of different regions of the protein.     

Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.     

Structural and mechanistic insights into the oxy form of tyrosinase from molecular dynamics simulations

The first, long time scale (16-ns) ligand field molecular dynamics (LFMD) simulations of the oxy form of tyrosinase are reported. The calculations use our existing type 3 copper force field for the peroxido-bridged [Cu₂O₂]²⁺ unit which is here translated from MMFF into the AMBER format together with a new charge scheme. The protein secondary and tertiary structures are not significantly altered by removing the ‘caddie’ protein, ORF378, which must be bound to tyrosinase before crystals will grow. A comprehensive principal component analysis of the Cartesian coordinates from the final 8 ns shows that the protein backbone is relatively rigid. However, the significant butterfly fold of the [Cu₂O₂]²⁺ moiety observed in the X-ray structure, presumably due to the caddie protein tyrosine at the active site, is absent in the simulations. LFMD gives a clear and persistent distinction between equatorial and axial Cu–N distances, with the latter about 0.2 Å longer and remaining syn to each other. However, the two coordination spheres display important differences. LFMD simulations of the symmetric model complex [μ-η²:μ²-O₂{Cu(Meim)₃}₂]²⁺ (Meim is 5-methyl-1H-imidazole) provide a mechanism for syn–anti interchange of axial ligands which suggests, in combination with the old experimental X-ray data, the new LFMD simulations and traditional coordination chemistry arguments, that His₅₄ on Cu_A is ‘insipiently axial’ and that a combination of a butterfly distortion of the [Cu₂O₂]²⁺ group and a rotation of the Cu_A(His)₃ moiety converts the vacant, initially axial, binding site on Cu_A into a much more favourable equatorial site.     

Binding of antifusion peptides with HIVgp41 from molecular dynamics simulations: quantitative correlation with experiment

Peptides based on C-terminal regions of the human immunodeficiency virus (HIV) viral protein gp41 represent an important new class of antiviral therapeutics called peptide fusion inhibitors. In this study, computational methods were used to model the binding of six peptides that contain residues that pack into a conserved hydrophobic pocket on HIVgp41, an attractive target site for the development of small molecule inhibitors. Free energies of binding were computed using molecular mechanics Generalized Born surface area (MM-GBSA) methods from molecular dynamics (MD) simulations, which employed either explicit (TIP3P) or continuum Generalized Born (GB) water models and strong correlations between experimental and computational affinities were obtained in both cases. Energy decomposition of the TIP3P-MD results (r2 = 0.75) reveals that variation in experimental affinity is highly correlated with changes in intermolecular van der Waals energies (deltaE(vdw)) on both a local (residue-based, r2 = 0.94) and global (peptide-based, r2 = 0.84) scale. The results show that differential association of C-peptides with HIVgp41 is driven solely by changes within the conserved pocket supporting the hypothesis that this region is an important drug target site. Such strong agreement with experiment is notable given the large size of the ligands (34 amino-acids) relative to the small range of experimental affinities (2 kcal/mol) and demonstrates good sensitivity of this computational method for simulating peptide fusion inhibitors. Finally, inspection of simulation trajectories identified a highly populated pi-type hydrogen bond, which formed between Gln575 on the receptor and the aromatic ring of peptide ligand Phe631, which could have important implications for drug design.     

Flexible fitting of high-resolution x-ray structures into cryoelectron microscopy maps using biased molecular dynamics simulations

A methodology for flexible fitting of all-atom high-resolution structures into low-resolution cryoelectron microscopy (cryo-EM) maps is presented. Flexibility of the modeled structure is simulated by classical molecular dynamics and an additional effective potential is introduced to enhance the fitting process. The additional potential is proportional to the correlation coefficient between the experimental cryo-EM map and a synthetic map generated for an all-atom structure being fitted to the map. The additional forces are calculated as a gradient of the correlation coefficient. During the molecular dynamics simulations under the additional forces, the molecule undergoes a conformational transition that maximizes the correlation coefficient, which results in a high-accuracy fit of all-atom structure into a cryo-EM map. Using five test proteins that exhibit structural rearrangement during their biological activity, we demonstrate performance of our method. We also test our method on the experimental cryo-EM of elongation factor G and show that the model obtained is comparable to previous studies. In addition, we show that overfitting can be avoided by assessing the quality of the fitted model in terms of correlation coefficient and secondary structure preservation.     

Mimicking the action of GroEL in molecular dynamics simulations: application to the refinement of protein structures

Bacterial chaperonin, GroEL, together with its co-chaperonin, GroES, facilitates the folding of a variety of polypeptides. Experiments suggest that GroEL stimulates protein folding by multiple cycles of binding and release. Misfolded proteins first bind to an exposed hydrophobic surface on GroEL. GroES then encapsulates the substrate and triggers its release into the central cavity of the GroEL/ES complex for folding. In this work, we investigate the possibility to facilitate protein folding in molecular dynamics simulations by mimicking the effects of GroEL/ES namely, repeated binding and release, together with spatial confinement. During the binding stage, the (metastable) partially folded proteins are allowed to attach spontaneously to a hydrophobic surface within the simulation box. This destabilizes the structures, which are then transferred into a spatially confined cavity for folding. The approach has been tested by attempting to refine protein structural models generated using the ROSETTA procedure for ab initio structure prediction. Dramatic improvements in regard to the deviation of protein models from the corresponding experimental structures were observed. The results suggest that the primary effects of the GroEL/ES system can be mimicked in a simple coarse-grained manner and be used to facilitate protein folding in molecular dynamics simulations. Furthermore, the results support the assumption that the spatial confinement in GroEL/ES assists the folding of encapsulated proteins.     

Effects of chain length on the aggregation of model polyglutamine peptides: molecular dynamics simulations

Aggregation in the brain of polyglutamine-containing proteins is either a cause or an associated symptom of nine hereditary neurodegenerative disorders including Huntington's disease. The molecular level mechanisms by which these proteins aggregate are still unclear. In an effort to shed light on this important phenomenon, we are investigating the aggregation of model polyglutamine peptides using molecular-level computer simulation with a simplified model of polyglutamine that we have developed. This model accounts for the most important types of intra- and inter-molecular interactions-hydrogen bonding and hydrophobic interactions-while allowing the folding process to be simulated in a reasonable time frame. The model is used to examine the folding of isolated polyglutamine peptides 16, 32, and 48 residues long and the folding and aggregation of systems of 24 model polyglutamine peptides 16, 24, 32, 36, 40, and 48 residues long. Although the isolated polyglutamine peptides did form some alpha and beta backbone-backbone hydrogen bonds they did not have as many of these bonds as they would have if they had folded into a complete alpha helix or beta sheet. In one of the simulations on the isolated polyglutamine peptide 48 residues long, we observed a structure that resembles a beta helix. In the multi-chain simulations we observed amorphous aggregates at low temperatures, ordered aggregates with significant beta sheet character at intermediate temperatures, and random coils at high temperatures. We have found that the temperature at which the model peptides undergo the transition from amorphous aggregates to ordered aggregates and the temperature at which the model peptides undergo the transition from ordered aggregates to random coils increase with increasing chain length. Our finding that the stability of the ordered aggregates increases as the peptide chain length increases may help to explain the experimentally observed relation between polyglutamine tract length and aggregation in vitro and disease progression in vivo. We have also observed in our simulations that the optimal temperature for the formation of beta sheets increases with chain length up to 36 glutamine residues but not beyond. Equivalently, at fixed temperature we find a transition from a region dominated by random coils at chain lengths less than 36 to a region dominated by relatively ordered beta sheet structures at chain lengths greater than 36. Our finding of this critical chain length of 36 glutamine residues is interesting because a critical chain length of 37 glutamine residues has been observed experimentally.     

Insights into molecular interactions between CaM and its inhibitors from molecular dynamics simulations and experimental data

In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca²⁺-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the “open” and “closed” conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM’s inhibitors correlated well with available experimental data as the r² obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca²⁺-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca²⁺-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca²⁺-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.     

Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations

The interfacial activation of many lipases at water/lipid interface is mediated by large conformational changes of a so‐called lid subdomain that covers up the enzyme active site. Here we investigated using molecular dynamic simulations in different explicit solvent environments (water, octane and water/octane interface) the molecular mechanism by which the lid motion of Burkholderia cepacia lipase might operate. Although B. cepacia lipase has so far only been crystallized in open conformation, this study reveals for the first time the major conformational rearrangements that the enzyme undergoes under the influence of the solvent, which either exposes or shields the active site from the substrate. In aqueous media, the lid switches from an open to a closed conformation while the reverse motion occurs in organic environment. In particular, the role of a subdomain facing the lid on B. cepacia lipase conformational rearrangements was investigated using position‐restrained MD simulations. Our conclusions indicate that the sole mobility of α9 helix side‐chains of B. cepacia lipase is required for the full completion of the lid conformational change which is essentially driven by α5 helix movement. The role of selected α5 hydrophobic residues on the lid movement was further examined. In silico mutations of two residues, V138 and F142, were shown to drastically modify the conformational behavior of B. cepacia lipase. Overall, our results provide valuable insight into the role played by the surrounding environment on the lid conformational rearrangement and the activation of B. cepacia lipase. Proteins 2009. © 2009 Wiley‐Liss, Inc.