Interpreting Dynamically-Averaged Scalar Couplings in Proteins

The experimental determination of scalar three-bond coupling constants represents a powerful method to probe both the structure and dynamics of proteins. The detailed structural interpretation of such coupling constants is usually based on Karplus relationships, which allow the measured couplings to be related to the torsion angles of the molecules. As the measured couplings are sensitive to thermal fluctuations, the parameters in the Karplus relationships are better derived from ensembles representing the distributions of dihedral angles present in solution, rather than from single conformations. We present a method to derive such parameters that uses ensembles of conformations determined through dynamic-ensemble refinement – a method that provides structural ensembles that simultaneously represent both the structure and the associated dynamics of a protein.     

Rotamer Dynamics: Analysis of Rotamers in Molecular Dynamics Simulations of Proteins

Given by χ torsional angles, rotamers describe the side-chain conformations of amino acid residues in a protein based on the rotational isomers (hence the word rotamer). Constructed rotamer libraries, based on either protein crystal structures or dynamics studies, are the tools for classifying rotamers (torsional angles) in a way that reflect their frequency in nature. Rotamer libraries are routinely used in structure modeling and evaluation. In this perspective article, we would like to encourage researchers to apply rotamer analyses beyond their traditional use. Molecular dynamics (MD) of proteins highlight the in silico behavior of molecules in solution and thus can identify favorable side-chain conformations. In this article, we used simple computational tools to study rotamer dynamics (RD) in MD simulations. First, we isolated each frame in the MD trajectories in separate Protein Data Bank files via the cpptraj module in AMBER. Then, we extracted torsional angles via the Bio3D module in R language. The classification of torsional angles was also done in R according to the penultimate rotamer library. RD analysis is useful for various applications such as protein folding, study of rotamer-rotamer relationship in protein-protein interaction, real-time correlation between secondary structures and rotamers, study of flexibility of side chains in binding site for molecular docking preparations, use of RD as guide in functional analysis and study of structural changes caused by mutations, providing parameters for improving coarse-grained MD accuracy and speed, and many others. Major challenges facing RD to emerge as a new scientific field involve the validation of results via easy, inexpensive wet-lab methods. This realm is yet to be explored.     

Correlation Spectroscopy and Molecular Dynamics Simulations to Study the Structural Features of Proteins

In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm²/s to 44±3 µm²/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm²/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm²/s and 31 µm²/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.     

Chemomechanical Regulation of SNARE Proteins Studied with Molecular Dynamics Simulations

SNAP-25B is a neuronal protein required for neurotransmitter (NT) release and is the target of Botulinum Toxins A and E. It has two SNARE domains that form a four-helix bundle when combined with syntaxin 1A and synaptobrevin. Formation of the three-protein complex requires both SNARE domains of SNAP-25B to align parallel, stretching out a central linker. The N-terminal of the linker has four cysteines within eight amino acids. Palmitoylation of these cysteines helps target SNAP-25B to the membrane; however, these cysteines are also an obvious target for oxidation, which has been shown to decrease SNARE complex formation and NT secretion. Because the linker is only slightly longer than the SNARE complex, formation of a disulfide bond between two cysteines might shorten it sufficiently to reduce secretion by limiting complex formation. To test this idea, we have carried out molecular dynamics simulations of the SNARE complex in the oxidized and reduced states. Indeed, marked conformational differences and a reduction of helical content in SNAP-25B upon oxidation are seen. Further differences are found for hydrophobic interactions at three locations, crucial for the helix-helix association. Removal of the linker induced different conformational changes than oxidation. The simulations suggest that oxidation of the cysteines leads to a dysfunctional SNARE complex, thus downregulating NT release during oxidative stress.     

Molecular Dynamics Study of Structure,Folding, and Aggregation of Poly-PR and Poly-GR Proteins

Poly-proline-arginine (poly-PR) and poly-glycine-arginine (poly-GR) proteins are believed to be the most toxic dipeptide repeat (DPR) proteins that are expressed by the hexanucleotide repeat expansion mutation in C9ORF72, which are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) diseases. Their structural information and mechanisms of toxicity remain incomplete, however. Using molecular dynamics simulation and all-atom model of proteins, we study folding and aggregation of both poly-PR and poly-GR. The results indicate formation of double-helix structure during the aggregation of poly-PR into dimers, whereas no stable aggregate is formed during the aggregation of poly-GR; the latter only folds into α-helix and double-helix structures that are similar to those formed in the folding of poly-glycine-alanine (poly-GA) protein. Our findings are consistent with the experimental data indicating that poly-PR and poly-GR are less likely to aggregate because of the hydrophilic arginine residues within their structures. Such characteristics could, however, in some respect facilitate migration of the DPR proteins between and within cells and, at the same time, give proline residues the benefits of activating the receptors that regulate ionotropic effect in neurons, resulting in death or malfunction of neurons because of the abnormal increase or decrease of the ion transmission. This may explain the neurotoxicities of poly-PR and poly-GR associated with many neurodegenerative diseases. To our knowledge, this is the first molecular dynamics simulation of the phenomena involving poly-PR and poly-GR proteins.     

Discrete Molecular Dynamics Can Predict Helical Prestructured Motifs in Disordered Proteins

Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available.     

Effects of Molecular Crowding on the Dynamics of Intrinsically Disordered Proteins

Inside cells, the concentration of macromolecules can reach up to 400 g/L. In such crowded environments, proteins are expected to behave differently than in vitro. It has been shown that the stability and the folding rate of a globular protein can be altered by the excluded volume effect produced by a high density of macromolecules. However, macromolecular crowding effects on intrinsically disordered proteins (IDPs) are less explored. These proteins can be extremely dynamic and potentially sample a wide ensemble of conformations under non-denaturing conditions. The dynamic properties of IDPs are intimately related to the timescale of conformational exchange within the ensemble, which govern target recognition and how these proteins function. In this work, we investigated the macromolecular crowding effects on the dynamics of several IDPs by measuring the NMR spin relaxation parameters of three disordered proteins (ProTα, TC1, and α-synuclein) with different extents of residual structures. To aid the interpretation of experimental results, we also performed an MD simulation of ProTα. Based on the MD analysis, a simple model to correlate the observed changes in relaxation rates to the alteration in protein motions under crowding conditions was proposed. Our results show that 1) IDPs remain at least partially disordered despite the presence of high concentration of other macromolecules, 2) the crowded environment has differential effects on the conformational propensity of distinct regions of an IDP, which may lead to selective stabilization of certain target-binding motifs, and 3) the segmental motions of IDPs on the nanosecond timescale are retained under crowded conditions. These findings strongly suggest that IDPs function as dynamic structural ensembles in cellular environments.     

Molecular Dynamics Study of Water in Hydrogels

Abstract

Molecular dynamics simulations are performed for aqueous solutions of polymers: Poly (vinyl alcohol) (PVA), Poly (vinyl methylether) (PVME), and Poly (N-isopropyl acrylamide) (PNiPAM). The distributions and dynamics of hydrogen-bonds, the translational diffusion of water, and the orientational relaxation of water are analyzed to investigate the properties of water which is highly influenced by the surrounding polymer chains. The water molecules around the polymer chains are highly hindered by the chains.     

Molecular Dynamics Simulations of Proteins in Water Without the Truncation of Long-range Coulomb Interactions

A new program package (COSMOS90) for molecular dynamics simulations was developed to simulate large molecular systems consisting of more than tens of thousands of atoms without the truncation of long-range coulomb interactions. This program package was based on a new approximation scheme (PPPC) for calculating efficiently the coulomb interactions without sacrificing accuracy. In this approximation scheme, the group of charges at a long distance from each atom was represented by a total charge and total dipole moment of the group. In order to assess the accuracy of PPPC and the ability of COSMOS90, molecular dynamics simulations were carried out for a large system consisting of 16108 atoms (human lysozyme in water) for 50 ps using this program package. The coulomb energy per solute atom was calculated with only five percent of the error found in the 10 Å cut-off approximation (about 0.9 kcal/mol versus 18 kcal/mol, respectively). The molecular dynamics simulations using COSMOS90 require no more CPU time than the simulations based on the 10 Å cut-off approximation of the conventional programs for macromolecular simulations.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

Arenavirus Dynamics in Experimentally and Naturally Infected Rodents

Infectious diseases of wildlife are typically studied using data on antibody and pathogen levels. In order to interpret these data, it is necessary to know the course of antibodies and pathogen levels after infection. Such data are typically collected using experimental infection studies in which host individuals are inoculated in the laboratory and sampled over an extended period, but because laboratory conditions are controlled and much less variable than natural conditions, the immune response and pathogen dynamics may differ. Here, we compared Morogoro arenavirus infection patterns between naturally and experimentally infected multimammate mice (Mastomys natalensis). Longitudinal samples were collected during three months of bi-weekly trapping in Morogoro, Tanzania, and antibody titer and viral RNA presence were determined. The time of infection was estimated from these data using a recently developed Bayesian approach, which allowed us to assess whether the natural temporal patterns match the previously observed patterns in the laboratory. A good match was found for 52% of naturally infected individuals, while most of the mismatches can be explained by the presence of chronically infected individuals (35%), maternal antibodies (10%), and an antibody detection limit (25%). These results suggest that while laboratory data are useful for interpreting field samples, there can still be differences due to conditions that were not tested in the laboratory.     

Molecular Dynamics Study of Nitrogen in Slit Micropores

Abstract

We present results of a computer simulation study of fluid nitrogen in model slit micropores. The model used for the micropore allows for the permeability of the pore wall to the confined fluid to be precisely controlled, while maintaining the atomic nature of the wall. Density and orientation profiles, wall permeabilities and diffusion coefficients have been obtained for systems with pore walls ranging from the almost impermeable to the completely permeable. Both the density and orientation profiles exhibit nonuniform behavior, while we observe anisotropy in the diffusion coefficients.     

Application of Correlated Residual Dipolar Couplings to the Determination of the Molecular Alignment Tensor Magnitude of Oriented Proteins and Nucleic Acids

Residual dipolar couplings (RDC) between nuclear spins in partially aligned samples offer unique insights into biomacromolecular structure and dynamics. To fully benefit from the RDC data, accurate knowledge of the magnitude ( D (a)) and rhombicity ( R ) of the molecular alignment tensor, A, is important. An extended histogram method (EHM) is presented which extracts these parameters more effectively from dipolar coupling data. The method exploits the correlated nature of RDCs for structural elements of planar geometry, such as the one-bond (13)C'(i)-(13)C(i)(alpha), (13)C'(i)-(15)N(i+1), and (15)N(i+1)-(1)H(N)(i+1) couplings in peptide bonds of proteins, or suitably chosen combinations of (1) D (C1'H1'), (1) D (C2'H2'), (1) D (C1'C2'), (2) D (C2'H1'), (2) D (C1'H2'), and (3) D (H1'H2') couplings in nucleic acids, to generate an arbitrarily large number of synthetic RDCs. These synthetic couplings result in substantially improved histograms and resulting values of D (a) and R, compared with histograms generated solely from the original sets of correlated RDCs, particularly when the number of planar fragments for which couplings are available is small. An alternative method, complementary to the EHM, is also described, which uses a systematic grid search procedure, based on least-squares fitting of sets of correlated RDCs to structural elements of known geometry, and provides an unambiguous lower limit for the degree of molecular alignment.     

The Role of Molecular Dynamics Simulations for the Study of Slow Dynamics

Abstract

A two step strategy is proposed to study dynamical properties of a physical system much slower than the time scales accessible by molecular dynamics simulations. The strategy is applied to investigate the slow dynamics of supercooled liquids.     

Molecular Dynamics simulations of Inhibitor of Apoptosis Proteins and identification of potential small molecule inhibitors

Chemotherapeutic resistance due to over expression of Inhibitor of Apoptosis Proteins (IAPs) XIAP, survivin and livin has been observed in various cancers. In the current study, Molecular Dynamics (MD) simulations were carried out for all three IAPs and a common ligand binding scaffold was identified. Further, a novel sequence based motif specific to these IAPs was designed. SMAC is an endogenous inhibitor of IAPs. Screening of ChemBank for compounds similar to lead SMAC-non-peptidomimetics yielded a cemadotin related compound NCIMech_000654. Cemadotin is a derivative of natural anti-tumor peptide dolastatin-15; hence these compounds were docked against all three IAPs. Based on our analysis, we propose that NCIMech_000654/dolastatin-15/cemadotin derivatives may be investigated for their potential in inhibiting XIAP, survivin and livin.     

Prediction of methyl-side Chain Dynamics in Proteins

A simple analytical model is presented for the prediction of methyl-side chain dynamics in comparison with S(2) order parameters obtained by NMR relaxation spectroscopy. The model, which is an extension of the local contact model for backbone order parameter prediction, uses a static 3D protein structure as input. It expresses the methyl-group S(2) order parameters as a function of local contacts of the methyl carbon with respect to the neighboring atoms in combination with the number of consecutive mobile dihedral angles between the methyl group and the protein backbone. For six out of seven proteins the prediction results are good when compared with experimentally determined methyl-group S(2) values with an average correlation coefficient r = 0.65+/-0.14. For the unusually rigid cytochrome c(2) no significant correlation between prediction and experiment is found. The presented model provides independent support for the reliability of current side-chain relaxation methods along with their interpretation by the model-free formalism.     

Interaction of Amyloid Inhibitor Proteins with Amyloid Beta Peptides: Insight from Molecular Dynamics Simulations

Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ) peptide aggregation is crucial for designing treatment for Alzheimer''s disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ_17–42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.     

A Molecular Dynamics Simulation Study of Coaxial Stacking in RNA

Abstract

We report on unrestrained molecular dynamics simulations of an RNA tetramer binding to a tetra-nucleotide overhang at the 5′-end of an RNA hairpin (nicked structure) and of the corresponding continuous hairpin with Na⁺ as counterions. The simulations lead to stable structures and in this way a structural model for the coaxially stacked RNA hairpin is generated. The stacking interface in the coaxially stacked nicked hairpin structure is characterized by a reduced twist and shift and a slightly increased propeller twist as compared to the continuous system. This leads to an increased overlap between C22 and G23 in the stacking interface of the nicked structure. In the simulations the continuous RNA hairpin has an almost straight helical axis. On the other hand, the corresponding axis for the nicked structure exhibits a marked kink of 39°. The stacking interface exhibits no increased flexibility as compared to the corresponding base pair step in the continuous structure.     

Constant-pH Molecular Dynamics Study of Kyotorphin in an Explicit Bilayer

To our knowledge, we present the first constant-pH molecular dynamics study of the neuropeptide kyotorphin in the presence of an explicit lipid bilayer. The overall conformation freedom of the peptide was found to be affected by the interaction with the membrane, in accordance with previous results using different methodologies. Analysis of the interactions between the N-terminus amine group of the peptide and several lipid atoms shows that the membrane is able to stabilize both ionized and neutral forms of kyotorphin, resulting in a pK_a value that is similar to the one obtained in water. This illustrates how a detailed molecular model of the membrane leads to rather different results than would be expected from simply regarding it as a low-dielectric slab.