Explicit Solvent Molecular Dynamics Research Articles

Mechanism of pKID/KIX Association Studied by Molecular Dynamics Free Energy Simulations.

The phosphorylated kinase-inducible domain (pKID) associates with the kinase interacting domain (KIX) via a coupled folding and binding mechanism. The pKID domain is intrinsically disordered when unbound and upon phosphorylation at Ser133 binds to the KIX domain adopting a well-defined kinked two-helix structure. In order to identify putative hot spot residues of binding that could serve as an initial stable anchor, we performed in silico alanine scanning free energy simulations. The simulations indicate that charged residues including the phosphorylated central Ser133 of pKID make significant contributions to binding. However, these are of slightly smaller magnitude compared to several hydrophobic side chains not defining a single dominant binding hot spot. Both continuous molecular dynamics (MD) simulations and free energy analysis demonstrate that phosphorylation significantly stabilizes the central kinked motif around Ser133 of pKID and shifts the conformational equilibrium toward the bound conformation already in the absence of KIX. This result supports a view that pKID/KIX association follows in part a conformational selection process. During a 1.5 μs explicit solvent MD simulation, folding of pKID on the surface of KIX was observed after an initial contact at the bound position of the phosphorylation site was enforced following a sequential process of αA helix association and a stepwise association and folding of the second αB helix compatible with available experimental results.

Dynamics simulation of soybean agglutinin (SBA) dimer reveals the impact of glycosylation on its enhanced structural stability

The legume lectins are widely used as a model system for studying protein–carbohydrate and protein–protein interactions. They exhibit a fascinating quaternary structure variation. Recently, it has become clear that lectins exist as oligomers. Soybean agglutinin is a tetrameric legume lectin, each of whose subunits are glycosylated. In the present study we explore the main origin for the stability of soybean agglutinin dimer. In order to understand the role of glycosylation on the dimeric interface, we have carried out normal (298K), high temperatures (380K, 500K) long explicit solvent molecular dynamics (MD) simulations and compared the structural and conformational changes between the glycosylated and non-glycosylated dimers. The study reveals that the high degree of stability at normal temperature is mostly contributed by interfacial ionic interactions (~200 kcal/mol) between polar residues like Lys, Arg, Asp, Thr, Ser, Asn and Gln (62%). It maintains its overall folded conformation due to high subunit interactions at the non-canonical interface. Mainly five important hydrogen bonds between CO of one β sheet of one subunit with the N-H of other β strand of the other subunit help to maintain the structural integrity. Ten inter subunit salt-bridge interactions between Arg 185–Asṕ192, Lys 163–Asṕ169, Asp 169–Lyś 163 and Asp 192–Arǵ 185 at non-canonical interface appear to be important to maintain the three dimensional structure of SBA dimer. Moreover, our simulation results revealed that increase in vibrational entropy could decrease the free energy and contribute to the glycan-induced stabilization by ~45 kcal/mol at normal temperature.

Structural and activity characterization of human PHPT1 after oxidative modification.

Phosphohistidine phosphatase 1 (PHPT1), the only known phosphohistidine phosphatase in mammals, regulates phosphohistidine levels of several proteins including those involved in signaling, lipid metabolism, and potassium ion transport. While the high-resolution structure of human PHPT1 (hPHPT1) is available and residues important for substrate binding and catalytic activity have been reported, little is known about post-translational modifications that modulate hPHPT1 activity. Here we characterize the structural and functional impact of hPHPT1 oxidation upon exposure to a reactive oxygen species, hydrogen peroxide (H2O2). Specifically, liquid chromatography-tandem mass spectrometry was used to quantify site-specific oxidation of redox-sensitive residues of hPHPT1. Results from this study revealed that H2O2 exposure induces selective oxidation of hPHPT1 at Met95, a residue within the substrate binding region. Explicit solvent molecular dynamics simulations, however, predict only a minor effect of Met95 oxidation in the structure and dynamics of the apo-state of the hPHPT1 catalytic site, suggesting that if Met95 oxidation alters hPHPT1 activity, then it will do so by altering the stability of an intermediate state. Employing a novel mass spectrometry-based assay, we determined that H2O2–induced oxidation does not impact hPHPT1 function negatively; a result contrary to the common conception that protein oxidation is typically a loss-of-function modification.

The Hinge Region Strengthens the Nonspecific Interaction between Lac-Repressor and DNA: A Computer Simulation Study.

LacI is commonly used as a model to study the protein-DNA interaction and gene regulation. The headpiece of the lac-repressor (LacI) protein is an ideal system for investigation of nonspecific binding of the whole LacI protein to DNA. The hinge region of the headpiece has been known to play a key role in the specific binding of LacI to DNA, whereas its role in nonspecific binding process has not been elucidated. Here, we report the results of explicit solvent molecular dynamics simulation and continuum electrostatic calculations suggesting that the hinge region strengthens the nonspecific interaction, accounting for up to 50% of the micro-dissociation free energy of LacI from DNA. Consequently, the rate of microscopic dissociation of LacI from DNA is reduced by 2~3 orders of magnitude in the absence of the hinge region. We find the hinge region makes an important contribution to the electrostatic energy, the salt dependence of electrostatic energy, and the number of salt ions excluded from binding of the LacI-DNA complex.

Thermodynamics, morphology, and kinetics of early-stage self-assembly of π-conjugated oligopeptides

Synthetic oligopeptides containing -conjugated cores self-assemble novel materials with attractive electronic and photophysical properties. All-atom, explicit solvent molecular dynamics simulations of Asp-Phe-Ala-Gly-OPV3-Gly-Ala-Phe-Asp peptides were used to parameterise an implicit solvent model to simulate early-stage self-assembly. Under low-pH conditions, peptides assemble into -sheet-like stacks with strongly favorable monomer association free energies of . Aggregation at high-pH produces disordered aggregates destabilised by Coulombic repulsion between negatively charged Asp termini (). In simulations of hundreds of monomers over 70 ns we observe the spontaneous formation of up to undecameric aggregates under low-pH conditions. Modeling assembly as a continuous-time Markov process, we infer transition rates between different aggregate sizes and microsecond relaxation times for early-stage assembly. Our data suggests a hierarchical model of assembly in which peptides coalesce into small clusters over tens of nanoseconds followed by structural ripening and diffusion limited aggregation on longer time scales. This work provides new molecular-level understanding of early-stage assembly, and a means to study the impact of peptide sequence and aromatic core chemistry upon the thermodynamics, assembly kinetics, and morphology of the supramolecular aggregates.

Base-Pair Level Analysis of DNA Four-Way Junction Structure and Dynamics

Immobile four-way junctions (4WJs) form a core structural motif of structured DNA assemblies. Understanding the sequence-level impact of four-way junctions on their structure, stability, and flexibility plays an important role in achieving Angstrom-level spatial control over nucleic acid nanodevices. 4WJs may exist in one of two stacked conformational isomers that are preferentially adopted when free in solution but forced into one or the other configuration in programmed multi-junction assemblies. It has been shown experimentally that the base sequences around the sites of strand crossover in these junctions have a significant impact on the conformational preferences of the two isomers.1,2 In particular, some sequences exhibit significant bias for one isomeric state, whereas others have equal preference between the two isomers. Here, we use explicit solvent and counterion molecular dynamics simulations to explore the base-pair level interactions that give rise to 4WJ conformational states using the classic Seeman J1 junction3 system as a point of reference. A pseudo-alchemical path approach is used to investigate how nicks placed at the site of the strand crossovers impact B-form DNA and crossover geometry and stability, and their dependence on core 4WJ sequence. Free energy calculations and analysis of the base-pair level degrees of freedom4 for each step along this path reveal the structural origin of the significant impact of base sequence on local structure, and suggest how these in turn impact global junction behaviour.

Oligomeric Structures of an Aggregation-Triggering Fragment of SOD1 Protein

Abstract: Amyloid fibril formation of copper-zinc superoxide dismutase (SOD1) is linked to familial amyotrophic lateral sclerosis (fALS), a progressive neurodegenerative disease. A recent experimental study has shown that 147GVIGIAQ153 peptide of the SOD1 C-terminal segment can form amyloid fibril and also accelerate the aggregation of full-length SOD1, while substitution of isoleucine at site 149 by proline blocks fibril formation of this peptide. However, the oligomeric structure and aggregation mechanism of this fragment remain elusive. In this study, we have investigated the hexameric structures of GVIGIAQ and its I149P variant by performing extensive atomistic explicit-solvent replica exchange molecular dynamics (REMD) simulations. We found that the GVIGIAQ hexamers adopt various β-sheet-rich conformations, including disordered aggregates and, to a much lesser extent, ordered fibril-like bilayer β-sheets and β-barrels. Structural analysis shows that four-stranded monolayer β-sheets are the smallest β-sheets that can form both bilayer β-sheets and β-barrels. In contrast, substitution of I149 by proline significantly reduces the β-sheet probability of all residues, especially those close to I149 in amino acid sequence, thus resulting in the vanish of bilayer β-sheets, a decrease of β-barrels, and a dramatic increase of disordered aggregates. The lack of bilayer β-sheets provides an explanation for the experimental observation of no fibril formation for I149P variant. MD simulation results on the structural stability of different sizes of experimental bilayer β-sheet fibril models further support the importance of I149 on fibril formation and also suggest that the bilayer β-sheet consisting of eight chains might be the smallest nucleus for 147GVIGIAQ153 fibril formation.

ErmBL Translation on the Ribosome in the Presence of Erythromycin is Stalled by Inhibition of Peptide Bond Formation

Translation of the Streptococcus sanguis ErmBL peptide is stalled in the presence of the antibiotic erythromycin, which binds inside the ribosomal exit tunnel. The stalling regulates the expression of the downstream gene of the resistance methyltransferase ErmB. Here, we investigate the stalling mechanism by explicit-solvent all-atom molecular dynamics simulations of the ribsome-ErmBL complex in the presence and absence of erythromycin. The simulations, totaling 12 µs simulation time, were started from a 3.6-A-resolution cryo-EM structure. In the cryo-EM structure the ribose of A76 of the peptidyl tRNA is shifted from its canonical pre-attack position. In the simulations without erythromycin, the ErmBL peptide explores conformations, which would overlap with the antibiotic. Together, these two findings show that erythromycin actively restrains the conformation of the peptide inside the tunnel and leads to the shift of the A76 ribose. Further, 23S nucleotides 2504-2506 and 2452 adopt different conformations depending on the presence of erythromycin. These nucleotides form a part of the A site cavity and are shifted away from the P site in the presence of erythromycin. In the stalled complex, the A-site tRNA carries a charged lysine which in the simulations, interacts with these nucleotides and follows their movement. This movement in turn, increases the distance between the attacking alpha-amino group of the lysine and the carbonyl-carbon of the peptide by more than 1 A, suggesting a lower rate of peptide bond formation. Upon mutation of the lysine to an alanine, we observe a decrease of this distance. This decrease is in agreement with the observation that the mutation alleviates stalling. On the basis of the results, we propose that a mutation of the lysine to an arginine, would be expected to preserve stalling.

Global and Local Conformational Heterogeniety Governs the Pre-Nucleation Phase in Amyloidogenic Self-Assembly

The formation of amyloid fibrils has been found to be a nucleation-dependent reaction for which the emergence of oligomeric entities is an inevitable and thus presumably, a critical intermediate step in the filamentous growth process. So far it has been not straight-forward to translate the extensive structural knowledge gained for the stable, fibrillar and crystalline states to the soluble oligomers prevalent during the pre-nucleation phase of the multi-step aggregation pathways. In this contribution we investigate the dynamical and structural properties of pre-brillar oligomeric states at atomistic resolution for several amyloidogenic peptide and protein fragments using unbiased explicit solvent molecular dynamics (MD) simulations. We have examined in particular the following questions: Which common secondary and teriary structural characteristics can be found in low molecular weight amyloid oligomers? What role do self-complentary side chain interfaces of intermolecular beta-sheets play in the oligomerization process? We analyzed simulations using multiple force fields in a consensus approach, in addition the results are compared in the light of recent experimental measurements done on model systems for peptide oligomerization. An extensive collision cross section analysis revealed heterogeneous oligomeric aggregates that lack a single, defined tertiary structure during the pre-nucleation phase. To gain insight into the structural organization of the early aggregates spontaneously formed on the microsecond time scale, we probed the substructure of the oligomers and found an increased ordering of intra-molecular conformations and inter-molecular packing motifs with growing aggregate size. We furthermore found that the ensemble of ordered higher-order oligomers sampled a multitude of common beta-structure motifs and was composed of emerging beta-sheet subunit layers. Our results suggest that distinct fibril-like conformations emerge in the oligomeric state and underscore the notion that certain pre-nucleated oligomers serve as a critical intermediate step on-pathway to fibrils.

Investigation of the pH Induced Conformational Rearrangement of Influenza Hemagglutinin

Influenza hemaggutinin (HA), a trimeric membrane fusion glycoprotein, guides the invasion of flu viruses into its host cells through endocytosis after binding to their receptors. During this process, a reduced endosomal pH triggers a large-scale structural rearrangement of HA2, the evolutionarily conserved stem domain of HA. This structural change is responsible for the fusion of the viral and the host membranes, facilitating the subsequent delivery of viral genome. Although the end states structures of the HA2 transition have been crystallized, the molecular details of this transition is unknown. To map out the overall picture, here we employ a combination of both coarse-grained and explicit solvent molecular dynamics simulations with the aim of understanding the underlying mechanism of membrane fusion. Our simulations suggest that the overall transition is separated into a fast phase and a slow phase. At the fast phase, HA2 experiences a fast order-disorder transition at a junction domain (Loop3-4) and reaches an asymmetric configuration. In the subsequent slow phase HA2 starts two transition pathways. Also, the results reveal that a reduction in pH destabilizes HA2 by protonating some key residues near its fusion peptides. Finally, a timescale competition among different domains of HA2 is shown to be necessary for HA function.

Mapping Allostery in the Two Domain Constructs of PDZ3 and SH3 with Artificial Domains

Synthetic fusion proteins of two or multiple domains using recombinant DNA technology are recently emerging in the field of protein engineering. The functional properties of the designed proteins were already enhanced and utilized for wide variety of biological and pharmaceutical applications [1]. However, there is no information about the super-molecular properties of two domain constructs of structurally and functionally different proteins. In order to understand the allostery at molecular level, we have performed all-atom explicit solvent molecular dynamics simulations in the isobaric-isothermal ensemble. We have selected the well characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ) along with other 5 artificial domains. Among other methods, we used IEM methodology to describe effect of second domain in the chimeric construct on the allostery of the first domain [2]. The influence of 5 artificial domains on the PDZ3 and SH3 were determined using a range of structural, dynamical and statistical analysis including residue fluctuation and inter-residue interaction energies in the solvent phase. Each amino acid contribution to the total intra-molecular stabilization energy of PDZ3 and SH3 were identified for all chimeras by means of direct and reverse combination approach. This allows us to describe artificial domains as modulators of dynamical and allosteric effect of the PDZ3 and SH3 domains.

Microsecond-Scale MD Simulations of HIV-1 DIS Kissing-Loop Complexes Predict Bulged-In Conformation of the Bulged Bases and Reveal Interesting Differences between Available Variants of the AMBER RNA Force Fields.

We report an extensive set of explicit solvent molecular dynamics (MD) simulations (∼25 μs of accumulated simulation time) of the RNA kissing-loop complex of the HIV-1 virus initiation dimerization site. Despite many structural investigations by X-ray, NMR, and MD techniques, the position of the bulged purines of the kissing complex has not been unambiguously resolved. The X-ray structures consistently show bulged-out positions of the unpaired bases, while several NMR studies show bulged-in conformations. The NMR studies are, however, mutually inconsistent regarding the exact orientations of the bases. The earlier simulation studies predicted the bulged-out conformation; however, this finding could have been biased by the short simulation time scales. Our microsecond-long simulations reveal that all unpaired bases of the kissing-loop complex stay preferably in the interior of the kissing-loop complex. The MD results are discussed in the context of the available experimental data and we suggest that both conformations are biochemically relevant. We also show that MD provides a quite satisfactory description of this RNA system, contrasting recent reports of unsatisfactory performance of the RNA force fields for smaller systems such as tetranucleotides and tetraloops. We explain this by the fact that the kissing complex is primarily stabilized by an extensive network of Watson-Crick interactions which are rather well described by the force fields. We tested several different sets of water/ion parameters but they all lead to consistent results. However, we demonstrate that a recently suggested modification of van der Waals interactions of the Cornell et al. force field deteriorates the description of the kissing complex by the loss of key stacking interactions stabilizing the interhelical junction and excessive hydrogen-bonding interactions.

Refinement of the Sugar-Phosphate Backbone Torsion Beta for AMBER Force Fields Improves the Description of Z- and B-DNA.

Z-DNA duplexes are a particularly complicated test case for current force fields. We performed a set of explicit solvent molecular dynamics (MD) simulations with various AMBER force field parametrizations including our recent refinements of the ε/ζ and glycosidic torsions. None of these force fields described the ZI/ZII and other backbone substates correctly, and all of them underpredicted the population of the important ZI substate. We show that this underprediction can be attributed to an inaccurate potential for the sugar-phosphate backbone torsion angle β. We suggest a refinement of this potential, β(OL1), which was derived using our recently introduced methodology that includes conformation-dependent solvation effects. The new potential significantly increases the stability of the dominant ZI backbone substate and improves the overall description of the Z-DNA backbone. It also has a positive (albeit small) impact on another important DNA form, the antiparallel guanine quadruplex (G-DNA), and improves the description of the canonical B-DNA backbone by increasing the population of BII backbone substates, providing a better agreement with experiment. We recommend using β(OL1) in combination with our previously introduced corrections, εζ(OL1) and χ(OL4), (the combination being named OL15) as a possible alternative to the current β torsion potential for more accurate modeling of nucleic acids.

Large-Scale Analysis of 48 DNA and 48 RNA Tetranucleotides Studied by 1 μs Explicit-Solvent Molecular Dynamics Simulations.

An understanding of how the conformational behavior of single-stranded DNAs and RNAs depend on sequence is likely to be important for attempts to rationalize the thermodynamics of nucleic acid folding. In an attempt to further our understanding of such sequence dependences, we report here the results of 192 (1 μs) explicit-solvent molecular dynamics (MD) simulations of 48 DNA and 48 RNA tetranucleotide sequences performed using recently reported modifications to the AMBER force field. Each tetranucleotide was simulated starting from two different conformations, a fully natively stacked and a completely unstacked conformation, and populations of the various possible base stacking arrangements were analyzed. The simulations indicate that, for both DNA and RNA, the populations of fully natively stacked conformations increase linearly with increasing numbers of purines in the sequence, while the conformational entropies, computed by two complementary methods, decrease. Despite the comparatively short simulation times, the computed free energies of stacking of the 16 possible combinations of bases in the middle of the sequences are found to be in good correspondence with values reported recently from simulations of dinucleoside monophosphates using the same force field. Finally, consistent with recent reports from other groups, non-native stacking interactions, i.e., between bases that are not adjacent in sequence, are shown to be a recurring feature of the simulations; in particular, stacking interactions of bases in a i:i+2 relationship are shown to occur significantly more frequently when the intervening base is a pyrimidine. Given that the high prevalence of non-native stacking interactions is thought to be unrealistic, it appears that further parametrization work will be required before accurate conformational descriptions of single-stranded nucleic acids can be obtained with current force fields.

Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition.

RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD) simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15, and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS) P-test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein.

Accurate Binding Free Energy Predictions in Fragment Optimization.

Predicting protein-ligand binding free energies is a central aim of computational structure-based drug design (SBDD)--improved accuracy in binding free energy predictions could significantly reduce costs and accelerate project timelines in lead discovery and optimization. The recent development and validation of advanced free energy calculation methods represents a major step toward this goal. Accurately predicting the relative binding free energy changes of modifications to ligands is especially valuable in the field of fragment-based drug design, since fragment screens tend to deliver initial hits of low binding affinity that require multiple rounds of synthesis to gain the requisite potency for a project. In this study, we show that a free energy perturbation protocol, FEP+, which was previously validated on drug-like lead compounds, is suitable for the calculation of relative binding strengths of fragment-sized compounds as well. We study several pharmaceutically relevant targets with a total of more than 90 fragments and find that the FEP+ methodology, which uses explicit solvent molecular dynamics and physics-based scoring with no parameters adjusted, can accurately predict relative fragment binding affinities. The calculations afford R(2)-values on average greater than 0.5 compared to experimental data and RMS errors of ca. 1.1 kcal/mol overall, demonstrating significant improvements over the docking and MM-GBSA methods tested in this work and indicating that FEP+ has the requisite predictive power to impact fragment-based affinity optimization projects.

Molecular insight into amyloid oligomer destabilizing mechanism of flavonoid derivative 2-(4′ benzyloxyphenyl)-3-hydroxy-chromen-4-one through docking and molecular dynamics simulations

Aggregation of amyloid peptide (Aβ) has been shown to be directly related to progression of Alzheimer’s disease (AD). Aβ is neurotoxic and its deposition and aggregation ultimately lead to cell death. In our previous work, we reported flavonoid derivative (compound 1) showing promising result in transgenic AD model of Drosophila. Compound 1 showed prevention of Aβ-induced neurotoxicity and neuroprotective efficacy in Drosophila system. However, mechanism of action of compound 1 and its effect on the amyloid is not known. We therefore performed molecular docking and atomistic, explicit-solvent molecular dynamics simulations to investigate the process of Aβ interaction, inhibition, and destabilizing mechanism. Results showed different preferred binding sites of compound 1 and good affinity toward the target. Through the course of 35 ns molecular dynamics simulation, conformations_5 of compound 1 intercalates into the hydrophobic core near the salt bridge and showed major structural changes as compared to other conformations. Compound 1 showed interference with the salt bridge and thus reducing the inter strand hydrogen bound network. This minimizes the side chain interaction between the chains A–B leading to disorder in oligomer. Contact map analysis of amino acid residues between chains A and B also showed lesser interaction with adjacent amino acids in the presence of compound 1 (conformations_5). The study provides an insight into how compound 1 interferes and disorders the Aβ peptide. These findings will further help to design better inhibitors for aggregation of the amyloid oligomer.

Role of water and steric constraints in the kinetics of cavity–ligand unbinding

A key factor influencing a drug's efficacy is its residence time in the binding pocket of the host protein. Using atomistic computer simulation to predict this residence time and the associated dissociation process is a desirable but extremely difficult task due to the long timescales involved. This gets further complicated by the presence of biophysical factors such as steric and solvation effects. In this work, we perform molecular dynamics (MD) simulations of the unbinding of a popular prototypical hydrophobic cavity-ligand system using a metadynamics-based approach that allows direct assessment of kinetic pathways and parameters. When constrained to move in an axial manner, the unbinding time is found to be on the order of 4,000 s. In accordance with previous studies, we find that the cavity must pass through a region of sharp wetting transition manifested by sudden and high fluctuations in solvent density. When we remove the steric constraints on ligand, the unbinding happens predominantly by an alternate pathway, where the unbinding becomes 20 times faster, and the sharp wetting transition instead becomes continuous. We validate the unbinding timescales from metadynamics through a Poisson analysis, and by comparison through detailed balance to binding timescale estimates from unbiased MD. This work demonstrates that enhanced sampling can be used to perform explicit solvent MD studies at timescales previously unattainable, to our knowledge, obtaining direct and reliable pictures of the underlying physiochemical factors including free energies and rate constants.

Anisotropic time-resolved solution X-ray scattering patterns from explicit-solvent molecular dynamics.

Time-resolved wide-angle X-ray scattering (TR-WAXS) is an emerging experimental technique used to track chemical reactions and conformational transitions of proteins in real time. Thanks to increased time resolution of the method, anisotropic TR-WAXS patterns were recently reported, which contain more structural information than isotropic patterns. So far, however, no method has been available to compute anisotropic WAXS patterns of biomolecules, thus limiting the structural interpretation. Here, we present a method to compute anisotropic TR-WAXS patterns from molecular dynamics simulations. The calculations accurately account for scattering of the hydration layer and for thermal fluctuations. For many photo-excitable proteins, given a low intensity of the excitation laser, the anisotropic pattern is described by two independent components: (i) an isotropic component, corresponding to common isotropic WAXS experiments and (ii) an anisotropic component depending on the orientation of the excitation dipole of the solute. We present a set of relations for the calculation of these two components from experimental scattering patterns. Notably, the isotropic component is not obtained by a uniform azimuthal average on the detector. The calculations are illustrated and validated by computing anisotropic WAXS patterns of a spheroidal protein model and of photoactive yellow protein. Effects due to saturated excitation at high intensities of the excitation laser are discussed, including opportunities to extract additional structural information by modulating the laser intensity.

ATP dependent NS3 helicase interaction with RNA: insights from molecular simulations.

Non-structural protein 3 (NS3) helicase from hepatitis C virus is an enzyme that unwinds and translocates along nucleic acids with an ATP-dependent mechanism and has a key role in the replication of the viral RNA. An inchworm-like mechanism for translocation has been proposed based on crystal structures and single molecule experiments. We here perform atomistic molecular dynamics in explicit solvent on the microsecond time scale of the available experimental structures. We also construct and simulate putative intermediates for the translocation process, and we perform non-equilibrium targeted simulations to estimate their relative stability. For each of the simulated structures we carefully characterize the available conformational space, the ligand binding pocket, and the RNA binding cleft. The analysis of the hydrogen bond network and of the non-equilibrium trajectories indicates an ATP-dependent stabilization of one of the protein conformers. Additionally, enthalpy calculations suggest that entropic effects might be crucial for the stabilization of the experimentally observed structures.