Conformational Energy Research Articles

Estimation of structure and stability of MurE ligase from Salmonella enterica serovar Typhi

MurE ligase catalyzes the assembly of peptide moiety, an essential component of bacterial cell wall. We have explored the conformational stability and unfolding equilibrium behaviour of the protein MurE ligase by determining the conformational free energy, entropy and enthalpy parameters under stress conditions. MurE from Salmonella enterica Serovar Typhi was cloned, expressed and purified. Conformational changes associated with increasing concentration of GdmCl- and urea-induced denaturation of MurE were monitored using Circular Dichroism (CD) and fluorescence spectroscopies. The secondary structural content of protein estimated by CD experiment is in close agreement with the predicted MurE ligase structure by homology modeling. Denaturant-induced transition curve was analyzed for thermodynamic parameters. Average values for MurE ligase of ΔGD0 = 3.13 kcal mol−1, m = 1.52 kcal mol−1 M−1 and Cm (=ΔGD0/m) = 2.05 M were calculated in the presence of GdmCl whereas in the case of urea these were ΔGD0 = 3.04 kcal mol−1, m = 1.20 kcal mol−1 M−1 and Cm (=ΔGD0/m) = 2.53 M. The observed superposition of normalized transition curve of two independent optical properties suggested that GdmCl- and urea-induced denaturation follow a two-state process.

Quantitative Assessment of the Energetics of Dopamine Translocation by Human Dopamine Transporter.

Computational evaluation of the energetics of substrate binding, transport, and release events of neurotransmitter transporters at the molecular level is a challenge, as the structural transitions of these membrane proteins involve coupled global and local changes that span time scales of several orders of magnitude, from nanoseconds to seconds. Here, we provide a quantitative assessment of the energetics of dopamine (DA) translocation through the human DA transporter (hDAT), using a combination of molecular modeling, simulation, and analysis tools. DA-binding and -unbinding events, which generally involve local configurational changes, are evaluated using free-energy perturbation or adaptive biasing force methods. The global transitions between the outward-facing state and the inward-facing state, on the other hand, require a dual-boost accelerated molecular dynamics simulation. We present results on DA-binding/unbinding energetics under different conditions, as well as the conformational energy landscape of hDAT in both DA-bound and -unbound states. The study provides a tractable method of approach for quantitative evaluation of substrate-binding energetics and efficient estimation of conformational energy landscape, in general.

Computation of Hemagglutinin Free Energy Difference by the Confinement Method.

Hemagglutinin (HA) mediates membrane fusion, a crucial step during influenza virus cell entry. How many HAs are needed for this process is still subject to debate. To aid in this discussion, the confinement free energy method was used to calculate the conformational free energy difference between the extended intermediate and postfusion state of HA. Special care was taken to comply with the general guidelines for free energy calculations, thereby obtaining convergence and demonstrating reliability of the results. The energy that one HA trimer contributes to fusion was found to be 34.2 ± 3.4kBT, similar to the known contributions from other fusion proteins. Although computationally expensive, the technique used is a promising tool for the further energetic characterization of fusion protein mechanisms. Knowledge of the energetic contributions per protein, and of conserved residues that are crucial for fusion, aids in the development of fusion inhibitors for antiviral drugs.

Comprehensive analysis of the catalytic and structural properties of a mu-class glutathione s-transferase from Fasciola gigantica

Glutathione S‒transferases (GSTs) play an important role in the detoxification of xenobiotics. They catalyze the nucleophilic addition of glutathione (GSH) to nonpolar compounds, rendering the products water-soluble. In the present study, we investigated the catalytic and structural properties of a mu-class GST from Fasciola gigantica (FgGST1). The purified recombinant FgGST1 formed a homodimer composed of 25 kDa subunit. Kinetic analysis revealed that FgGST1 displays broad substrate specificity and shows high GSH conjugation activity toward 1-chloro-2,4-dinitrobenzene, 4-nitroquinoline-1-oxide, and trans-4-phenyl-3-butene-2-one and peroxidase activity towards trans-2-nonenal and hexa-2,4-dienal. The FgGST1 was highly sensitive to inhibition by cibacron blue. The cofactor (GSH) and inhibitor (cibacron blue) were docked, and binding sites were identified. The molecular dynamics studies and principal component analysis indicated the stability of the systems and the collective motions, respectively. Unfolding studies suggest that FgGST1 is a highly cooperative molecule because, during GdnHCl-induced denaturation, a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed. The protein is stabilized with a conformational free energy of about 10 ± 0.3 kcal mol−1. Additionally, the presence of conserved Pro-53 and structural motifs such as N-capping box and hydrophobic staple, further aided in the stability and proper folding of FgGST1.

Neural Network Based Prediction of Conformational Free Energies - A New Route toward Coarse-Grained Simulation Models.

Coarse-grained (CG) simulation models have become very popular tools to study complex molecular systems with great computational efficiency on length and time scales that are inaccessible to simulations at atomistic resolution. In so-called bottom-up coarse-graining strategies, the interactions in the CG model are devised such that an accurate representation of an atomistic sampling of configurational phase space is achieved. This means the coarse-graining methods use the underlying multibody potential of mean force (i.e., free-energy surface) derived from the atomistic simulation as parametrization target. Here, we present a new method where a neural network (NN) is used to extract high-dimensional free energy surfaces (FES) from molecular dynamics (MD) simulation trajectories. These FES are used for simulations on a CG level of resolution. The method is applied to simulating homo-oligo-peptides (oligo-glutamic-acid (oligo-glu) and oligo-aspartic-acid (oligo-asp)) of different lengths. We show that the NN not only is able to correctly describe the free-energy surface for oligomer lengths that it was trained on but also is able to predict the conformational sampling of longer chains.

Insight into partial agonism by observing multiple equilibria for ligand-bound and Gs-mimetic nanobody-bound \u03b21-adrenergic receptor

A complex conformational energy landscape determines G-protein-coupled receptor (GPCR) signalling via intracellular binding partners (IBPs), e.g., Gs and β-arrestin. Using 13C methyl methionine NMR for the β1-adrenergic receptor, we identify ligand efficacy-dependent equilibria between an inactive and pre-active state and, in complex with Gs-mimetic nanobody, between more and less active ternary complexes. Formation of a basal activity complex through ligand-free nanobody–receptor interaction reveals structural differences on the cytoplasmic receptor side compared to the full agonist-bound nanobody-coupled form, suggesting that ligand-induced variations in G-protein interaction underpin partial agonism. Significant differences in receptor dynamics are observed ranging from rigid nanobody-coupled states to extensive μs-to-ms timescale dynamics when bound to a full agonist. We suggest that the mobility of the full agonist-bound form primes the GPCR to couple to IBPs. On formation of the ternary complex, ligand efficacy determines the quality of the interaction between the rigidified receptor and an IBP and consequently the signalling level.

Role of electrostatic interactions in determining the G-quadruplex structures

We investigate the energetics of the antiparallel, hybrid and parallel type G-quadruplex structures of the human telomere DNA sequence. We find that both the conformational energy and solvation free energy of these structures are roughly inversely proportional to their radii of gyration. We rationalize this finding in terms of the dominance of the electrostatic contributions. We also show that the solvation free energy is more significant than the conformational energy in determining the G-quadruplex structures, which is in contrast to the canonical B-DNA structures. Our work will contribute to an understanding of the molecular mechanisms dictating various G-quadruplex topologies.

Free Energy of a Folded Polymer under Cylindrical Confinement

Monte Carlo computer simulations are used to study the conformational free energy of a folded polymer confined to a long cylindrical tube. The polymer is modeled as a hard-sphere chain. Its conformational free energy $F$ is measured as a function of $\lambda$, the end-to-end distance of the polymer. In the case of a flexible linear polymer, $F(\lambda)$ is a linear function in the folded regime with a gradient that scales as $f\equiv |dF/d\lambda| \sim N^0 D^{-1.20\pm 0.01}$ for a tube of diameter $D$ and a polymer of length $N$. This is close to the prediction $f \sim N^0 D^{-1}$ obtained from simple scaling arguments. The discrepancy is due in part to finite-size effects associated with the de-Gennes blob model. A similar discrepancy was observed for the folding of a single arm of a three-arm star polymer. We also examine backfolding of a semiflexible polymer of persistence length $P$ in the classic Odijk regime. In the overlap regime, the derivative scales $f \sim N^0 D^{-1.72\pm 0.02} P^{-0.35\pm 0.01}$, which is close to the prediction $f \sim N^0 D^{-5/3} P^{-1/3}$ obtained from a scaling argument that treats interactions between deflection segments at the second virial level. In addition, the measured free energy cost of forming a hairpin turn is quantitatively consistent with a recent theoretical calculation. Finally, we examine the scaling of $F(\lambda)$ for a confined semiflexible chain in the presence of an S-loop composed of two hairpins. While the predicted scaling of the free energy gradient is the same as that for a single hairpin, we observe a scaling of $f \sim D^{-1.91\pm 0.03} P^{-0.36\pm 0.01}$. Thus, the quantitative discrepancy between this measurement and the predicted scaling is somewhat greater for S-loops than for single hairpins.

Determination of Protein ps-ns Motions by High-Resolution Relaxometry.

Many of the functions of biomacromolecules can be rationalized by the characterization of their conformational energy landscapes: the structures of the dominant states, transitions between states and motions within states. Nuclear magnetic resonance (NMR) spectroscopy is the technique of choice to study internal motions in proteins. The determination of motions on picosecond to nanosecond timescales requires the measurement of nuclear spin relaxation rates at multiple magnetic fields. High sensitivity and resolution are obtained only at high magnetic fields, so that, until recently, site-specific relaxation rates in biomolecules were only measured over a narrow range of high magnetic fields. This limitation was particularly striking for the quantification of motions on nanosecond timescales, close to the correlation time for overall rotational diffusion. High-resolution relaxometry is an emerging technique to investigate picosecond-nanosecond motions of proteins. This approach uses a high-field NMR spectrometer equipped with a sample shuttle device, which allows for the measurement of the relaxation rate constants at low magnetic fields, while preserving the sensitivity and resolution of a high-field NMR spectrometer. The combined analysis of high-resolution relaxometry and standard high-field relaxation data provides a more accurate description of the dynamics of proteins, in particular in the nanosecond range. The purpose of this chapter is to describe how to perform high-resolution relaxometry experiments and how to analyze the rates measured with this technique.

Experimental Demonstration of a Sizeable Nonclassical CH···G Hydrogen Bond in Cyclohexane Derivatives: Stabilization of an Axial Cyano Group.

Base-catalyzed equilibration of anancomeric cyanocyclohexanes demonstrates that replacement of cis-3,5-dimethyl holding groups with electron-withdrawing CF3 groups dramatically increases the proportion of the axial cyano isomer present at equilibrium. The CF3 groups exert an effect on the conformational energy of the cyano group worth about 0.6 kcal/mol. A nonclassical hydrogen bond between the axial CN group and the syn-axial hydrogens is a major contributor to the axial stability of the group.

Conformational dynamics and free energy of BHRF1 binding to Bim BH3

The interaction between the Bim BH3 peptide and the viral protein BHRF1 is pivotal to understanding the fundamental molecular details of the mechanism used by the Epstein-Barr virus to trick the mammalian immune system. Here, we study the mechanism of binding/unbinding and compute the free energy for the association of the Bim peptide to the BHRF1 protein. Key elements of the binding mechanism are the conformational rearrangement together with a main free energy barrier of 11.5kcal/mol. The simulations show complete unbinding and rebinding of the Bim peptide to BHRF1. The peptide slowly dissociates, disrupting the hydrophobic contacts, then tilting to one side. The peptide then completely disrupts all the remaining interactions and moves into the bulk solvent. The rebinding of the peptide from the solvent to the receptor binding site occurs slowly. This is because the helix partially unfolds in the unbound state. Rebinding involves an intermediate state, in which the peptide interacts with the hydrophobic binding pocket, which mainly involves Leu 62, Arg 63, Ile 65, and Phe 69. This novel intermediate structure forms 65 contacts with the receptor before the peptide again reaches the bound state. The standard binding free energy value is close to the experimental Kd in the nanomolar range. Finally, we observe how the breathing motions of α3-α4 are coupled with the binding/unbinding of the Bim BH3 peptide. The structure of the intermediate can be used for designing novel peptide inhibitors of the BHRF1 protein.

Calculation of conformational free energies by confinement simulations in explicit water with implicit desolvation

The confinement method is a robust and conceptually simple free energy simulation method that allows the calculation of conformational free energy differences between highly dissimilar states. Application of the method to explicitly solvated systems requires a multi-stage simulation protocol for the calculation of desolvation free energies. Here we show that these desolvation free energies can be readily obtained from an implicit treatment, which is simpler and less costly. The accuracy and robustness of this protocol was shown by the calculation of conformational free energy differences of a series of explicitly solvated test systems. Given the accuracy and ease by which these free energy differences were obtained, the confinement method is promising for the treatment of conformational changes in large and complex systems.

Additive Driven Increase in Donor–Acceptor Copolymer Coupling Studied by X-ray Resonant Photoemission

The role of additives and thermal treatment in the formation of donor–acceptor copolymer organic films of PFO–DBT (poly[2,7-(9,9-dioctylfluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2,1,3-thiadiazole]) with increased transport properties is addressed by resonant Auger photoemission and core-hole clock spectroscopy, which allows an analysis of the competition between the inner shell core-hole lifetime and the motion of photoexcited electrons on a femtosecond time scale. From the branching of the competing core-hole decay channels, we study the delocalization dynamics of excited electrons over empty molecular orbitals. We find evidence of ultrafast charge-carrier transfer from specific orbitals (LUMO+1) and increased coupling in copolymer assemblies when a solvent additive (1,8 diiodooctane) is added and samples are post-treated with thermal annealing. Relative conformational energies and core-hole spectra were calculated by time-dependent density functional theory.

Hoisting-Loop in Bacterial Multidrug Exporter AcrB Is a Highly Flexible Hinge That Enables the Large Motion of the Subdomains.

The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as Escherichia coli's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cβ15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.

The Origin of Chalcogen-Bonding Interactions.

Favorable molecular interactions between group 16 elements have been implicated in catalysis, biological processes, and materials and medicinal chemistry. Such interactions have since become known as chalcogen bonds by analogy to hydrogen and halogen bonds. Although the prevalence and applications of chalcogen-bonding interactions continues to develop, debate still surrounds the energetic significance and physicochemical origins of this class of σ-hole interaction. Here, synthetic molecular balances were used to perform a quantitative experimental investigation of chalcogen-bonding interactions. Over 160 experimental conformational free energies were measured in 13 different solvents to examine the energetics of O···S, O···Se, S···S, O···HC, and S···HC contacts and the associated substituent and solvent effects. The strongest chalcogen-bonding interactions were found to be at least as strong as conventional H-bonds, but unlike H-bonds, surprisingly independent of the solvent. The independence of the conformational free energies on solvent polarity, polarizability, and H-bonding characteristics showed that electrostatic, solvophobic, and van der Waals dispersion forces did not account for the observed experimental trends. Instead, a quantitative relationship between the experimental conformational free energies and computed molecular orbital energies was consistent with the chalcogen-bonding interactions being dominated by n → σ* orbital delocalization between a lone pair (n) of a (thio)amide donor and the antibonding σ* orbital of an acceptor thiophene or selenophene. Interestingly, stabilization was manifested through the same acceptor molecular orbital irrespective of whether a direct chalcogen···chalcogen or chalcogen···H-C contact was made. Our results underline the importance of often-overlooked orbital delocalization effects in conformational control and molecular recognition phenomena.

Stability of Unfolded and Folded Protein Structures Using a 3D-RISM with the RMDFT.

Protein stability is determined by the characteristics of the protein itself as well as the surrounding solvent. Herein, we discuss the stability of the folded and unfolded structures of proteins obtained from Anton's long simulations (Lindorff-Larsen, K.; Piana, S.; Dror, R. O.; Shaw, D.E. Science, 2011, 334, 517-520). Specifically, the stabilities of CLN025, the WW domain variant GTT, the triple mutant of the redesigned protein G variant NuG2, and the de novo-designed three-helix bundle protein are investigated. The solvation free energy of the structures is calculated using the three-dimensional reference interaction site model with the reference-modified density functional theory. The total energy is given by the sum of the conformational energy and the solvation free energy, and their balance results in the stabilization of protein structure, as demonstrated by the correspondence between structures with the lowest total energy of all proteins to their native structures. Overall, these findings indicate that the total energy function is appropriate for evaluating the stability of protein folding systems. Moreover, decomposing the energy terms reveals that proteins achieve their stabilities from the balance between the conformational energy and the solvation free energy. In particular, the solvation entropy is the main contributor to the process of folding from more extended structures to compact structures. The native structure is more stable than the compact structure owing to competition between intramolecular and intermolecular interactions.

Ab-initio conformational epitope structure prediction using genetic algorithm and SVM for vaccine design

Background and objectiveT-cell epitope structure identification is a significant challenging immunoinformatic problem within epitope-based vaccine design. Epitopes or antigenic peptides are a set of amino acids that bind with the Major Histocompatibility Complex (MHC) molecules. The aim of this process is presented by Antigen Presenting Cells to be inspected by T-cells. MHC-molecule-binding epitopes are responsible for triggering the immune response to antigens. The epitope's three-dimensional (3D) molecular structure (i.e., tertiary structure) reflects its proper function. Therefore, the identification of MHC class-II epitopes structure is a significant step towards epitope-based vaccine design and understanding of the immune system. MethodsIn this paper, we propose a new technique using a Genetic Algorithm for Predicting the Epitope Structure (GAPES), to predict the structure of MHC class-II epitopes based on their sequence. The proposed Elitist-based genetic algorithm for predicting the epitope's tertiary structure is based on Ab-Initio Empirical Conformational Energy Program for Peptides (ECEPP) Force Field Model. The developed secondary structure prediction technique relies on Ramachandran Plot. We used two alignment algorithms: the ROSS alignment and TM-Score alignment. We applied four different alignment approaches to calculate the similarity scores of the dataset under test. We utilized the support vector machine (SVM) classifier as an evaluation of the prediction performance. ResultsThe prediction accuracy and the Area Under Receiver Operating Characteristic (ROC) Curve (AUC) were calculated as measures of performance. The calculations are performed on twelve similarity-reduced datasets of the Immune Epitope Data Base (IEDB) and a large dataset of peptide-binding affinities to HLA-DRB1*0101. The results showed that GAPES was reliable and very accurate. We achieved an average prediction accuracy of 93.50% and an average AUC of 0.974 in the IEDB dataset. Also, we achieved an accuracy of 95.125% and an AUC of 0.987 on the HLA-DRB1*0101 allele of the Wang benchmark dataset. ConclusionsThe results indicate that the proposed prediction technique “GAPES” is a promising technique that will help researchers and scientists to predict the protein structure and it will assist them in the intelligent design of new epitope-based vaccines.

Combined complete active space configuration interaction and perturbation theory applied to conformational energy prototypes: Rotation and inversion barriers

A second-order multireference perturbation theory, termed as IVO-SSMRPT which allows the use of CASCI reference wave functions with improved virtual orbitals (IVO) for capturing static correlation and state-specific parameterization of the state-universal electronic wave function in an attempt to account for dynamic correlation has been utilized in an investigation of the torsional properties of ethylene, silaethylene, hydrogen peroxide, hydrazine, and oxalyl chloride. We also calculate the barrier to inversion of ammonia. IVO-SSMRPT is robust and useful to scan energy surfaces as it avoids the intruder-state problem, a troubling aspect of various established MRPT methods, without exploiting level-shifting or increasing the size of the active space. We find that IVO-SSMRPT with the use of a relatively small active space and basis set can be compared with recent reference estimates which are reproduced within the expected precision indicating the method is useful for the study of rotation and inversion barriers of challenging molecules.

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch.

S-adenosyl-L-methionine (SAM) ligand binding induces major structural changes in SAM-I riboswitches, through which gene expression is regulated via transcription termination. Little is known about the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I riboswitch. Therefore, we have explored its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling analysis. We resolved four conformational states both in the presence and the absence of SAM and determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds coexist, and SAM binding only gradually shifts the populations toward terminator states. We observed a pronounced acceleration of conformational transitions upon SAM binding, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.

Thermodynamic projection of the antibody interaction network: The fountain energy landscape of molecular interaction systems

The adaptive humoral immune system of vertebrates functions by evolving a huge repertoire of binding proteins, which target potentially all molecules that come into contact with developing B cells. The key to endowing these binders with immunological activity is the adjustment of antibody structure and affinity against molecular targets. As a result, antibodies with a wide range of affinities and specificities evolve during the lifetime of an individual. I recently developed a quantitative model for the description of antibody homeostasis and suggested that a quantitative network can describe the dynamic antibody-antigen interaction space. Here, I project this molecular interaction space onto an energy landscape defined by conformational entropy and free energy of binding. I introduce the concept of binding fountain energy landscape, which allows the thermodynamic representation of binding events and paths of multiple interactions. I further show that the hypersurface of the binding fountain corresponds to the antibody-antigen interaction network. I propose that thymus independent and thymus dependent antibody responses show distinct patterns of changes in the energy landscape. Overall, the fountain energy landscape concept of molecular interactions allows a systems biological, thermodynamic perception and description of the functioning of the clonal humoral immune system.