Physics-Based Potentials for the Coupling between Backbone- and Side-Chain-Local Conformational States in the United Residue (UNRES) Force Field for Protein Simulations

The UNited RESidue (UNRES) model of polypeptide chains is a coarse-grained model in which each amino-acid residue is reduced to two interaction sites, namely, a united peptide group (p) located halfway between the two neighboring α-carbon atoms (C^αs), which serve only as geometrical points, and a united side chain (SC) attached to the respective C^α. Owing to this simplification, millisecond molecular dynamics simulations of large systems can be performed. While UNRES predicts overall folds well, it reproduces the details of local chain conformation with lower accuracy. Recently, we implemented new knowledge-based torsional potentials (Krupa et al. <i>J. Chem. Theory Comput.</i> <b>2013</b>, <i>9</i>, 4620–4632) that depend on the virtual-bond dihedral angles involving side chains: C^α···C^α···C^α···SC (τ⁽¹⁾), SC···C^α···C^α···C^α (τ⁽²⁾), and SC···C^α···C^α···SC (τ⁽³⁾) in the UNRES force field. These potentials resulted in significant improvement of the simulated structures, especially in the loop regions. In this work, we introduce the physics-based counterparts of these potentials, which we derived from the all-atom energy surfaces of terminally blocked amino-acid residues by Boltzmann integration over the angles λ⁽¹⁾ and λ⁽²⁾ for rotation about the C^α···C^α virtual-bond angles and over the side-chain angles χ. The energy surfaces were, in turn, calculated by using the semiempirical AM1 method of molecular quantum mechanics. Entropy contribution was evaluated with use of the harmonic approximation from Hessian matrices. One-dimensional Fourier series in the respective virtual-bond-dihedral angles were fitted to the calculated potentials, and these expressions have been implemented in the UNRES force field. Basic calibration of the UNRES force field with the new potentials was carried out with eight training proteins, by selecting the optimal weight of the new energy terms and reducing the weight of the regular torsional terms. The force field was subsequently benchmarked with a set of 22 proteins not used in the calibration. The new potentials result in a decrease of the root-mean-square deviation of the average conformation from the respective experimental structure by 0.86 Å on average; however, improvement of up to 5 Å was observed for some proteins

Role of the sulfur to α-carbon thioether bridges in thurincin H

<p>Thurincin H is a small protein produced by <i>Bacillus thuringiensis</i> SF361 with gram-positive antimicrobial properties. The toxins produced by <i>B. thuringiensis</i> are widely used in the agriculture as, e.g. natural preservatives in dairy products. The structure of thurincin H possesses four covalent sulfur to -carbon bonds that involve the cysteine side-chains; these bonds are probably responsible for the shape and stability of the protein and, thereby, for its antimicrobial properties. To examine the influence of the formation of the sulfur-carbon bonds on the folding pathways and stability of the protein, a series of canonical and multiplexed replica-exchange simulations with the coarse-grained UNRES force field was carried out without and with distance restraints imposed on selected S-C atom pairs. It was found that the order of the formation and breaking of the S-C thioether bonds significantly impacts on the foldability and stability of the thurincin H. It was also observed that thioether bridges play a major role in stabilizing the global fold of the protein, although it significantly diminishes the entropy of the system. The maximum foldability of thurincin H was observed in the presence of the optimal set of three out of four thioether bridges. Thus, the results suggest that the presence of ThnB enzyme and other agents that catalyze the formation of thioether bridges can be essential for correct folding of thurincin H and that the formation of the fourth bridge does not seem to facilitate folding; instead, it seems to rigidify the loop and prevent proteolysis.</p

Prediction of Protein Structure by Template-Based Modeling Combined with the UNRES Force Field

A new approach to the prediction of protein structures that uses distance and backbone virtual-bond dihedral angle restraints derived from template-based models and simulations with the united residue (UNRES) force field is proposed. The approach combines the accuracy and reliability of template-based methods for the segments of the target sequence with high similarity to those having known structures with the ability of UNRES to pack the domains correctly. Multiplexed replica-exchange molecular dynamics with restraints derived from template-based models of a given target, in which each restraint is weighted according to the accuracy of the prediction of the corresponding section of the molecule, is used to search the conformational space, and the weighted histogram analysis method and cluster analysis are applied to determine the families of the most probable conformations, from which candidate predictions are selected. To test the capability of the method to recover template-based models from restraints, five single-domain proteins with structures that have been well-predicted by template-based methods were used; it was found that the resulting structures were of the same quality as the best of the original models. To assess whether the new approach can improve template-based predictions with incorrectly predicted domain packing, four such targets were selected from the CASP10 targets; for three of them the new approach resulted in significantly better predictions compared with the original template-based models. The new approach can be used to predict the structures of proteins for which good templates can be found for sections of the sequence or an overall good template can be found for the entire sequence but the prediction quality is remarkably weaker in putative domain-linker regions

Use of Restraints from Consensus Fragments of Multiple Server Models To Enhance Protein-Structure Prediction Capability of the UNRES Force Field

Recently, we developed a new approach to protein-structure prediction, which combines template-based modeling with the physics-based coarse-grained UNited RESidue (UNRES) force field. In this approach, restrained multiplexed replica exchange molecular dynamics simulations with UNRES, with the C^α-distance and virtual-bond-dihedral-angle restraints derived from knowledge-based models are carried out. In this work, we report a test of this approach in the 11th Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP11), in which we used the template-based models from early-stage predictions by the LEE group CASP11 server (group 038, called “nns”), and further improvement of the method. The quality of the models obtained in CASP11 was better than that resulting from unrestrained UNRES simulations; however, the obtained models were generally worse than the final nns models. Calculations with the final nns models, performed after CASP11, resulted in substantial improvement, especially for multi-domain proteins. Based on these results, we modified the procedure by deriving restraints from models from multiple servers, in this study the four top-performing servers in CASP11 (nns, BAKER-ROSETTA­SERVER, Zhang-server, and QUARK), and implementing either all restraints or only the restraints on the fragments that appear similar in the majority of models (the <i>consensus fragments</i>), outlier models discarded. Tests with 29 CASP11 human-prediction targets with length less than 400 amino-acid residues demonstrated that the consensus-fragment approach gave better results, i.e., lower α-carbon root-mean-square deviation from the experimental structures, higher template modeling score, and global distance test total score values than the best of the parent server models. Apart from global improvement (repacking and improving the orientation of domains and other substructures), improvement was also reached for template-based modeling targets, indicating that the approach has refinement capacity. Therefore, the consensus-fragment analysis is able to remove lower-quality models and poor-quality parts of the models without knowing the experimental structure

Improvement of the Treatment of Loop Structures in the UNRES Force Field by Inclusion of Coupling between Backbone- and Side-Chain-Local Conformational States

The UNited RESidue (UNRES) coarse-grained model of polypeptide chains, developed in our laboratory, enables us to carry out millisecond-scale molecular-dynamics simulations of large proteins effectively. It performs well in <i>ab initio</i> predictions of protein structure, as demonstrated in the last Community Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP10). However, the resolution of the simulated structure is too coarse, especially in loop regions, which results from insufficient specificity of the model of local interactions. To improve the representation of local interactions, in this work, we introduced new side-chain-backbone correlation potentials, derived from a statistical analysis of loop regions of 4585 proteins. To obtain sufficient statistics, we reduced the set of amino-acid-residue types to five groups, derived in our earlier work on structurally optimized reduced alphabets [Solis, A. D.; Rackovsky, S. <i>Proteins: Struct., Func., Bioinf.</i>, <b>2000</b>, <i>38,</i> 149–164], based on a statistical analysis of the properties of amino-acid structures. The new correlation potentials are expressed as one-dimensional Fourier series in the virtual-bond-dihedral angles involving side-chain centroids. The weight of these new terms was determined by a trial-and-error method, in which Multiplexed Replica Exchange Molecular Dynamics (MREMD) simulations were run on selected test proteins. The best average root-mean-square deviations (RMSDs) of the calculated structures from the experimental structures below the folding-transition temperatures were obtained with the weight of the new side-chain-backbone correlation potentials equal to 0.57. The resulting conformational ensembles were analyzed in detail by using the Weighted Histogram Analysis Method (WHAM) and Ward’s minimum-variance clustering. This analysis showed that the RMSDs from the experimental structures dropped by 0.5 Å on average, compared to simulations without the new terms, and the deviation of individual residues in the loop region of the computed structures from their counterparts in the experimental structures (after optimum superposition of the calculated and experimental structure) decreased by up to 8 Å. Consequently, the new terms improve the representation of local structure

Cytochrome <i>c</i> oxidation activities of membranes from different <i>B</i>. <i>subtilis</i> strains.

<p>Cytochrome <i>c</i> oxidation activities of membranes from different <i>B</i>. <i>subtilis</i> strains.</p

HP0377 truncated variants act as wild type HP0377.

<p><b>(A)<i>In vitro</i> isomerase activity assay</b>. The reaction contained 40 μM scrambled RNase in 200 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 20 μM DTT, and 9 mM cCMP. The reaction was performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0377 and its variants. The cleavage of cCMP by refolded RNase was monitored continuously at 296 nm. The changes in the absorbance at 296 nm as a function of time are presented. Three independent experiments were performed. <b>(B) <i>In vitro</i> reductase assay of HP0377 variants towards apocytochrome c (HP1227)</b>. Two different SDS PAGEs were run to better visualize the shift between oxidized and reduced forms of proteins. Different redox forms were detected by nonreducing SDS-PAGE after AMS treatment, which results in an increase in the molecular mass of reduced proteins by about 0.5 kDa per thiol group. <b>(C) Glutaraldehyde crosslinking of truncated versions of HP0377. 1) N-terminal-shortened HP0377; 2) C-terminal-truncated HP0377</b>. Purified HP0377 truncated versions at 2.5 mg/ml were cross-linked in the presence of different concentration of glutaraldehyde: 1) purified HP0377 protein, 2) 0.001%, 3) 0.005%, 4) 0.01%, 5) 0.05%, 6) 0.1% glutaraldehyde. M—monomers, D—dimers.</p

Analysis of HP0377-apocytochrome c complex formation.

<p><b>(A) Size exclusion profile of the HP0377, apocytochrome, HP0377-apocytochrome complex and HP0377-apocytochrome complex treated with DTT separated on an Enrich</b>^™<b>sec70 column (Biorad) and monitored by absorbance at 280 nm</b>. HP0377 elutes as a peak at 10.7 min, with an estimated mass of 23.5 kDa, consistent with the size of the monomer. Apocytochrome elutes at 12.9 min, with estimated mass of 9.5 kDa. Complex eluted at 8.9 min, with estimated mass of 56 kDa. Complex treated with DTT elutes as two peaks at 12.9 min and 10.7 min, consistent with the sizes of the HP0377 and apocytochrome. The column was calibrated with Gel Filtration Standard (Bio-Rad): Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), Myoglobin (17 kDa), Vitamin B12 (1.35 kDa). The relative positions of the chosen standards are marked with arrows. <b>(B) SDS-PAGE analysis of HP0377-apocytochrome complex formation</b>. Complexes formed between HP0377_CSYA and apocytochrome were first purified using Ni-NTA resin, and then separated on a size exclusion column. The samples were next analyzed by SDS-PAGE with or without DTT.</p

Plots of the fluctuations of the α-carbon atoms as obtained by the UNRES simulation.

<p>The secondary-structure elements are shown above the abscissa. The black, thin line corresponds to the β factors as obtained from the HP0377 (PDB: 4FYC) structure. The simulation results are marked as follows: wild-type HP0377 (black “WT”); CSYC, LcP, (red, “LcP”); CSYC, TcT, (green, “TcT”). E95 and Q95 are colored blue and purple, respectively.</p

Primers used in this study.

<p>Primers used in this study.</p