Conformational transitions of the quercetin molecule via the rotations of its rings: a comprehensive theoretical study

Abstract

Quercetin is an important flavonoid compound, usually extracted from plants, vegetables and fruits such as blueberries, apples, green tea, wine, onions and possessing broad range of pharmacological properties, in particular, powerful antioxidant, antitoxic, antiinflammation and antimicrobial effects due to its various reactive sites. The structure of this phenolic compound consists of three (A?+?C) and B rings, bearing five hydroxyl groups. Primarily, the chemical structure of quercetin determines its physico-chemical properties. Earlier, it was established that isolated quercetin molecule can acquire 48 stable conformations (24 planar and 24 non-planar) due to the mobility of its hydroxyl groups and (A?+?C) and B rings with relative Gibbs free energies in the range of 0.0–25.3?kcal·mol^?1 under normal conditions (Brovarets’ et al., 2019c Brovarets’, O. O., & Hovorun, D. M. (2019c). Conformational diversity of the quercetin molecule: A quantum-chemical view. Journal of Biomolecular Structure and Dynamics. doi: 10.1080/07391102.2019.1645734[Taylor & Francis Online], [Web of Science ®] , [Google Scholar]). In this work by quantum-mechanical calculations at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory and Bader’s ‘Quantum Theory of Atoms in Molecules’, we have theoretically modeled the interconversions in the 24 pairs of the conformers of the quercetin molecule, occuring via the rotation of its non-deformable (A+С) and B rings around the С2-С1' bond through the quasi-orthogonal transition state with low values of the imaginary frequencies (28–33/29–36?cm^?1) and Gibbs free energies of activation in the range of 2.17–5.68/1.86–4.90?kcal·mol^?1 in the continuum with dielectric permittivity ε?=?1/ε?=?4 under normal conditions. Also, we studied the changes of the number of physico-chemical characteristics of all intramolecular-specific contacts – hydrogen bonds and attractive van der Waals contacts during these conformational rearrangements.

Communicated by Ramaswamy H. Sarma     

A new era of the prototropic tautomerism of the quercetin molecule: A QM/QTAIM computational advances

Abstract

In this study for the first time we have revealed and investigated in details 123 different prototropic tautomers of the most stable conformer of the quercetin molecule using quantum-mechanical calculations at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of QM theory. We have found that in the most energetically favorable prototropic tautomer mobile hydrogen atoms are localized at the О3, О3′, О4′, О5, and О7 exocyclic oxygen atoms. Molecular tautomers are in the range of the Gibbs free energies from 0.0 to 69.8?kcal·mol^?1, while zwitterionic ones – from 30.1 до 172.8?kcal·mol^?1 at normal conditions. It was also reliably established that the weakest point causing the decyclization of the molecule is its C ring – this reaction is launched by the transition of the proton from the C8H group to the endocyclic O1 oxygen atom. All prototropic tautomers, except two cases, are joined by the intramolecular cooperative specific interactions (from 1 to 5) – H-bonds and attractive van der Waals contacts, which have been revealed and characterized by QTAIM analysis.

Communicated by Ramaswamy H. Sarma     

Exploring the nature of the H-bonds between the human class II MHC protein,HLA-DR1 (DRB*0101) and the influenza virus hemagglutinin peptide,HA306-318, using the quantum theory of atoms in molecules

The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HC_aliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) >> P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.     

Atoms-in-molecules study of the genetically encoded amino acids. III. Bond and atomic properties and their correlations with experiment including mutation-induced changes in protein stability and genetic coding

This article presents a study of the molecular charge distributions of the genetically encoded amino acids (AA), one that builds on the previous determination of their equilibrium geometries and the demonstrated transferability of their common geometrical parameters. The properties of the charge distributions are characterized and given quantitative expression in terms of the bond and atomic properties determined within the quantum theory of atoms-in-molecules (QTAIM) that defines atoms and bonds in terms of the observable charge density. The properties so defined are demonstrated to be remarkably transferable, a reflection of the underlying transferability of the charge distributions of the main chain and other groups common to the AA. The use of the atomic properties in obtaining an understanding of the biological functions of the AA, whether free or bound in a polypeptide, is demonstrated by the excellent statistical correlations they yield with experimental physicochemical properties. A property of the AA side chains of particular importance is the charge separation index (CSI), a quantity previously defined as the sum of the magnitudes of the atomic charges and which measures the degree of separation of positive and negative charges in the side chain of interest. The CSI values provide a correlation with the measured free energies of transfer of capped side chain analogues, from the vapor phase to aqueous solution, yielding a linear regression equation with r2 = 0.94. The atomic volume is defined by the van der Waals isodensity surface and it, together with the CSI, which accounts for the electrostriction of the solvent, yield a linear regression (r2 = 0.98) with the measured partial molar volumes of the AAs. The changes in free energies of transfer from octanol to water upon interchanging 153 pairs of AAs and from cyclohexane to water upon interchanging 190 pairs of AAs, were modeled using only three calculated parameters (representing electrostatic and volume contributions) yielding linear regressions with r2 values of 0.78 and 0.89, respectively. These results are a prelude to the single-site mutation-induced changes in the stabilities of two typical proteins: ubiquitin and staphylococcal nuclease. Strong quadratic correlations (r2 approximately 0.9) were obtained between DeltaCSI upon mutation and each of the two terms DeltaDeltaH and TDeltaDeltaS taken from recent and accurate differential scanning calorimetry experiments on ubiquitin. When the two terms are summed to yield DeltaDeltaG, the quadratic terms nearly cancel, and the result is a simple linear fit between DeltaDeltaG and DeltaCSI with r2 = 0.88. As another example, the change in the stability of staphylococcal nuclease upon mutation has been fitted linearly (r2 = 0.83) to the sum of a DeltaCSI term and a term representing the change in the van der Waals volume of the side chains upon mutation. The suggested correlation of the polarity of the side chain with the second letter of the AA triplet genetic codon is given concrete expression in a classification of the side chains in terms of their CSI values and their group dipole moments. For example, all amino acids with a pyrimidine base as their second letter in mRNA possess side-chain CSI < or = 2.8 (with the exception of Cys), whereas all those with CSI > 2.8 possess an purine base. The article concludes with two proposals for measuring and predicting molecular complementarity: van der Waals complementarity expressed in terms of the van der Waals isodensity surface and Lewis complementarity expressed in terms of the local charge concentrations and depletions defined by the topology of the Laplacian of the electron density. A display of the experimentally accessible Laplacian distribution for a folded protein would offer a clear picture of the operation of the "stereochemical code" proposed as the determinant in the folding process.     

The investigation of the G-quadruplex aptamer selectivity to Pb2+ ion: a joint molecular dynamics simulation and density functional theory study

Abstract

The aptamers with the ability to form a G-quadruplex structure can be stable in the presence of some ions. Hence, study of the interactions between such aptamers and ions can be beneficial to determine the highest selective aptamer toward an ion. In this article, molecular dynamics (MD) simulations and quantum mechanics (QM) calculations have been applied to investigate the selectivity of the T30695 aptamer toward Pb²⁺ in comparison with some ions. The Free Energy Landscape (FEL) analysis indicates that Pb²⁺ has remained inside the aptamer during the MD simulation, while the other ions have left it. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) binding energies prove that the conformational stability of the aptamer is the highest in the presence of Pb²⁺. According to the compaction parameters, the greatest compressed ion-aptamer complex, and hence, the highest ion-aptamer interaction have been induced in the presence of Pb²⁺. The contact maps clarify the closer contacts between the nucleotides of the aptamer in the presence of Pb²⁺. The density functional theory (DFT) results show that Pb²⁺ forms the most stable complex with the aptamer, which is consistent with the MD results. The QM calculations reveal that the N-H bonds and the O…H distances are the longest and the shortest, respectively, in the presence of Pb²⁺. The obtained results verify that the strongest hydrogen bonds (HBs), and hence, the most compressed aptamer structure are induced by Pb²⁺. Besides, atoms in molecules (AIM) and natural bond orbital (NBO) analyses confirm the results.

Communicated by Ramaswamy H. Sarma     

Molecular modeling,dynamics studies and density functional theory approaches to identify potential inhibitors of SIRT4 protein from Homo sapiens : a novel target for the treatment of type 2 diabetes

Type 2 diabetes is one of the biggest health challenges in the world and WHO projects it to be the 7th leading cause of death in 2030. It is a chronic condition affecting the way our body metabolizes sugar. Insulin resistance is high risk factor marked by expression of Lipoprotein Lipases and Peroxisome Proliferator-Activated Receptor that predisposes to type 2 diabetes. AMP-dependent protein kinase in AMPK signaling pathway is a central sensor of energy status. Deregulation of AMPK signaling leads to inflammation, oxidative stress, and deactivation of autophagy which are implicated in pathogenesis of insulin resistance. SIRT4 protein deactivates AMPK as well as directly inhibits insulin secretion. SIRT4 overexpression leads to dyslipidimeia, decreased fatty acid oxidation, and lipogenesis which are the characteristic features of insulin resistance promoting type 2 diabetes. This makes SIRT4 a novel therapeutic target to control type 2 diabetes. Virtual screening and molecular docking studies were performed to obtain potential ligands. To further optimize the geometry of protein–ligand complexes Quantum Polarized Ligand Docking was performed. Binding Free Energy was calculated for the top three ligand molecules. In view of exploring the stereoelectronic features of the ligand, density functional theory approach was implemented at B3LYP/6-31G* level. 30 ns MD simulation studies of the protein–ligand complexes were done. The present research work proposes ZINC12421989 as potential inhibitor of SIRT4 with docking score (?7.54 kcal/mol), docking energy (?51.34 kcal/mol), binding free energy (?70.21 kcal/mol), and comparatively low energy gap (?0.1786 eV) for HOMO and LUMO indicating reactivity of the lead molecule.     

Toward a general mixed quantum/classical method for the calculation of the vibronic ECD of a flexible dye molecule with different stable conformers: Revisiting the case of 2,2,2‐trifluoro‐anthrylethanol

We extend a recently proposed mixed quantum/classical method for computing the vibronic electronic circular dichroism (ECD) spectrum of molecules with different conformers, to cases where more than one hindered rotation is present. The method generalizes the standard procedure, based on the simple Boltzmann average of the vibronic spectra of the stable conformers, and includes the contribution of structures that sample all the accessible conformational space. It is applied to the simulation of the ECD spectrum of (S)‐2,2,2‐trifluoroanthrylethanol, a molecule with easily interconvertible conformers, whose spectrum exhibits a pattern of alternating positive and negative vibronic peaks. Results are in very good agreement with experiment and show that spectra averaged over all the sampled conformational space can deviate significantly from the simple average of the contributions of the stable conformers. The present mixed quantum/classical method is able to capture the effect of the nonlinear dependence of the rotatory strength on the molecular structure and of the anharmonic couplings among the modes responsible for molecular flexibility. Despite its computational cost, the procedure is still affordable and promises to be useful in all cases where the ECD shape arises from a subtle balance between vibronic effects and conformational variety.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Improving the stability of an antibody variable fragment by a combination of knowledge-based approaches: validation and mechanisms

Numerous approaches have been described to obtain variable fragments of antibodies (Fv or scFv) that are sufficiently stable for their applications. Here, we combined several knowledge-based methods to increase the stability of pre-existing scFvs by design. Firstly, the consensus sequence approach was used in a non-stringent way to predict a large basic set of potentially stabilizing mutations. These mutations were then prioritized by other methods of design, mainly the formation of additional hydrogen bonds, an increase in the hydrophilicity of solvent exposed residues, and previously described mutations in other antibodies. We validated this combined method with antibody mAbD1.3, directed against lysozyme. Fourteen potentially stabilizing mutations were designed and introduced into scFvD1.3 by site-directed mutagenesis, either individually or in combinations. We characterized the effects of the mutations on the thermodynamic stability of scFvD1.3 by experiments of unfolding with urea, monitored by spectrofluorometry, and tested the additivity of their effects by double-mutant cycles. We also quantified the individual contributions of the resistance to denaturation ([urea](1/2)) and cooperativity of unfolding (m) to the variations of stability and the energy of coupling between mutations by a novel approach. Most mutations (75%) were stabilizing and none was destabilizing. The progressive recombination of the mutations into the same molecule of scFvD1.3 showed that their effects were mostly additive or synergistic, provided a large overall increase in protein stability (9.1 kcal/mol), and resulted in a highly stable scFvD1.3 derivative. The mechanisms of the mutations and of their combinations involved variations in the resistance to denaturation, cooperativity of unfolding, and likely residual structures of the denatured state, which was constrained by two disulfide bonds. This combined method should be applicable to any recombinant antibody fragment, through a single step of mutagenesis.     

Molecular dynamics study of the primary charge separation reactions in Photosystem I: Effect of the replacement of the axial ligands to the electron acceptor A0

Molecular dynamics (MD) calculations, a semi-continuum (SC) approach, and quantum chemistry (QC) calculations were employed together to investigate the molecular mechanics of ultrafast charge separation reactions in Photosystem I (PS I) of Thermosynechococcus elongatus. A molecular model of PS I was developed with the aim to relate the atomic structure with electron transfer events in the two branches of cofactors. A structural flexibility map of PS I was constructed based on MD simulations, which demonstrated its rigid hydrophobic core and more flexible peripheral regions. The MD model permitted the study of atomic movements (dielectric polarization) in response to primary and secondary charge separations, while QC calculations were used to estimate the direct chemical effect of the A_0A/A_0B ligands (Met or Asn in the 688/668 position) on the redox potential of chlorophylls A_0A/A_0B and phylloquinones A_1A/A_1B. A combination of MD and SC approaches was used to estimate reorganization energies λ of the primary (λ₁) and secondary (λ₂) charge separation reactions, which were found to be independent of the active branch of electron transfer; in PS I from the wild type, λ₁ was estimated to be 390 ± 20 mV, while λ₂ was estimated to be higher at 445 ± 15 mV. MD and QC approaches were used to describe the effect of substituting Met688_PsaA/Met668_PsaB by Asn688_PsaA/Asn668_PsaB on the energetics of electron transfer. Unlike Met, which has limited degrees of freedom in the site, Asn was found to switch between two relatively stable conformations depending on cofactor charge. The introduction of Asn and its conformation flexibility significantly affected the reorganization energy of charge separation and the redox potentials of chlorophylls A_0A/A_0B and phylloquinones A_1A/A_1B, which may explain the experimentally observed slowdown of secondary electron transfer in the M688N_PsaA variant. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Chromophore Structure of Cyanobacterial Phytochrome Cph1 in the Pr State: Reconciling Structural and Spectroscopic Data by QM/MM Calculations

A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His²⁶⁰ in the chromophore-binding pocket such that either the δ-nitrogen (M-HSD) or the ɛ-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His²⁶⁰ with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.Abbreviations: Agp1, phytochrome from Agrobacterium tumefaciens; α-CPC, α-subunit of C-phycocyanin; BV, biliverdin IXα; B3LYP, three-parameter exchange functional according to Becke, Lee, Yang, and Parr; DFT, density functional theory; DrBphP, phytochrome from Deinococcus radiodurans; GAF, domain found in cGMP-specific phosphodiesterases; MM, molecular mechanics; MD, molecular dynamics; N-H ip, N-H in-plane bending; PCB, phycocyanobilin; PED, potential energy distribution; phyA, plant phytochrome; Pr, Pfr, red- and far-red absorbing parent states of phytochrome; PΦB, phytochromobilin; QM, quantum mechanics; RMSD, root mean-square deviation; RR, resonance Raman     

Consequences of Domain Insertion on the Stability and Folding Mechanism of a Protein

SlyD, the sensitive-to-lysis protein from Escherichia coli, consists of two domains. They are not arranged successively along the protein chain, but one domain, the “insert-in-flap” (IF) domain, is inserted internally as a guest into a surface loop of the host domain, which is a prolyl isomerase of the FK506 binding protein (FKBP) type. We used SlyD as a model to elucidate how such a domain insertion affects the stability and folding mechanism of the host and the guest domain. For these studies, the two-domain protein was compared with a single-domain variant SlyDΔIF, SlyD* without the chaperone domain (residues 1-69 and 130-165) in which the IF domain was removed and replaced by a short loop, as present in human FKBP12. Equilibrium unfolding and folding kinetics followed an apparent two-state mechanism in the absence and in the presence of the IF domain. The inserted domain decreased, however, the stability of the host domain in the transition region and decelerated its refolding reaction by about 10-fold. This originates from the interruption of the chain connectivity by the IF domain and its inherent instability. To monitor folding processes in this domain selectively, a Trp residue was introduced as fluorescent probe. Kinetic double-mixing experiments revealed that, in intact SlyD, the IF domain folds and unfolds about 1000-fold more rapidly than the FKBP domain, and that it is strongly stabilized when linked with the folded FKBP domain. The unfolding limbs of the kinetic chevrons of SlyD show a strong downward curvature. This deviation from linearity is not caused by a transition-state movement, as often assumed, but by the accumulation of a silent unfolding intermediate at high denaturant concentrations. In this kinetic intermediate, the FKBP domain is still folded, whereas the IF domain is already unfolded.