Angle Χ1 Research Articles

Searching conformational analysis of Asp residues through theoretical 3J vicinal coupling constants and Karplus equations

AbstractThe dependence of the vicinal spin–spin coupling constants on the torsion side chain angle χ1 through the Karplus equations is considered for the study of the structure of Asp amino acid residues. Experimental and theoretical, obtained with density functional theory methods, vicinal coupling constants combined with extended Karplus equations, which include six coefficients, are applied to a dipeptide model of the amino acid Asp. To find out the empirical χ1 angles of the side chain, a statistical analysis procedure is developed to compute the rmsd values and find the χ1 as the minimum of those values. The χ1 values obtained in this work for nine Asp residues of the flavodoxin protein Desulfovibrio vulgaris are successfully compared with those derived by nuclear magnetic resonance and X‐rays.

Flap Dynamics in Pepsin-Like Aspartic Proteases: A Computational Perspective Using Plasmepsin-II and BACE-1 as Model Systems.

The flexibility of β hairpin structure known as the flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequential similarity. In this study, we have used apo Plm-II and BACE-1 as model systems. In the apo form of the proteases, a conserved tyrosine residue in the flap region remains in a dynamic equilibrium between the normal and flipped states through rotation of the χ1 and χ2 angles. Independent MD simulations of Plm-II and BACE-1 remained stuck either in the normal or flipped state. Metadynamics simulations using side-chain torsion angles (χ1 and χ2 of tyrosine) as collective variables sampled the transition between the normal and flipped states. Qualitatively, the two states were predicted to be equally populated. The normal and flipped states were stabilized by H-bond interactions to a tryptophan residue and to the catalytic aspartate, respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine, reduced the flexibility of the flap and resulted in a flap collapse (flap loses flexibility and remains stuck in a particular state). This is in accordance with previous experimental studies, which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Our results suggest that the ring flipping associated with the tyrosine side-chain is the key order parameter that governs flap dynamics and opening of the binding pocket in most pepsin-like aspartic proteases.

Conformational change due to replacement of disulfide with selenosulfide and diselenide in dipeptide vicinal cysteine loop

Replacement of disulfide bridges with diselenide bridges is increasingly common to improve the stability, foldability, and structural refinement of the cysteine-rich polypeptides. Even though the global structural features are similar due to the replacement of disulfide with diselenide, the local conformational differences have been reported in a few polypeptides. The current report has used the constrained vicinal cysteine disulfide as the model to access the influence of the replacement of disulfides with diselenide on the local conformations. Using the density functional theory (DFT), structures of dipeptide vicinal loops are optimized by systematically replacing sulfur with selenium. Conformations of the disulfide/selenosulfide/diselenide were identified using the side-chain torsional angle χ1, χ2, χ3, χ2′, χ1′ and mapped to one of the possible 32 conformations of the cysteine disulfide. Further, the influence of the change of configuration of Cα-atom of cysteine/selenocysteine from ‘R′ to ‘S′ configuration and peptide bond from cis to trans has also been accessed on the conformations of dipeptide vicinal loops. The results indicate that diselenide/selenosulfide explores additional conformational space apart from accommodating the conformations observed in the vicinal disulfide which is more amplified in the heterochiral system. Differences have been observed at the internal coordinates of the optimized structures of dipeptide vicinal disulfide, selenosulfide, and diselenide. The change in free energy (ΔG), spin density (Δs(r)), and electron density (Δρ(r)) was also calculated due to the replacement of disulfide with selenosulfide/diselenide. Conformational analysis of disulfides and that of the replaced diselenides in the crystal structures of the proteins retrieved from PDB have also indicated the retention as well as differences in the local conformations. The tendency of the diselenide loop to explore the additional conformational space may prompt for the local conformational differences in the corresponding disulfide to diselenide replaced polypeptides.

Computational Protocol to Evaluate Side-Chain Vicinal Spin-Spin Coupling Constants and Karplus Equation in Amino Acids: Alanine Dipeptide Model.

A computational protocol has been applied to the alanine dipeptide model in order to study the side-chain conformation, the calculated spin-spin coupling constants involved in the side-chain χ1 angle, and theoretical extended Karplus equations developed for amino acids. Two structures within the backbone secondary conformation are used to predict coupling constants which in addition are employed to analyze the effect of those structures on the resulting Karplus extended equations. The number of Fourier coefficients to be included within the Karplus equations is critically analyzed. Wave function and density functional methods as well as different basis sets are compared to find appropriate Karplus coefficients in the most efficient way. The influence of exchange and correlation functionals and the solvent effect on the calculated couplings are considered.

Crystallographic Structures of IlvN·Val/Ile Complexes: Conformational Selectivity for Feedback Inhibition of Aceto Hydroxy Acid Synthases.

Conformational factors that predicate selectivity for valine or isoleucine binding to IlvN leading to the regulation of aceto hydroxy acid synthase I (AHAS I) of Escherichia coli have been determined for the first time from high-resolution (1.9-2.43 Å) crystal structures of IlvN·Val and IlvN·Ile complexes. The valine and isoleucine ligand binding pockets are located at the dimer interface. In the IlvN·Ile complex, among residues in the binding pocket, the side chain of Cys43 is 2-fold disordered (χ1 angles of gauche- and trans). Only one conformation can be observed for the identical residue in the IlvN·Val complexes. In a reversal, the side chain of His53, located at the surface of the protein, exhibits two conformations in the IlvN·Val complex. The concerted conformational switch in the side chains of Cys43 and His53 may play an important role in the regulation of the AHAS I holoenzyme activity. A significant result is the establishment of the subunit composition in the AHAS I holoenzyme by analytical ultracentrifugation. Solution nuclear magnetic resonance and analytical ultracentrifugation experiments have also provided important insights into the hydrodynamic properties of IlvN in the ligand-free and -bound states. The structural and biophysical data unequivocally establish the molecular basis for differential binding of the ligands to IlvN and a rationale for the resistance of IlvM to feedback inhibition by the branched-chain amino acids.

Inherent versus induced protein flexibility: Comparisons within and between apo and holo structures.

Understanding how ligand binding influences protein flexibility is important, especially in rational drug design. Protein flexibility upon ligand binding is analyzed herein using 305 proteins with 2369 crystal structures with ligands (holo) and 1679 without (apo). Each protein has at least two apo and two holo structures for analysis. The inherent variation in structures with and without ligands is first established as a baseline. This baseline is then compared to the change in conformation in going from the apo to holo states to probe induced flexibility. The inherent backbone flexibility across the apo structures is roughly the same as the variation across holo structures. The induced backbone flexibility across apo-holo pairs is larger than that of the apo or holo states, but the increase in RMSD is less than 0.5 Å. Analysis of χ1 angles revealed a distinctly different pattern with significant influences seen for ligand binding on side-chain conformations in the binding site. Within the apo and holo states themselves, the variation of the χ1 angles is the same. However, the data combining both apo and holo states show significant displacements. Upon ligand binding, χ1 angles are frequently pushed to new orientations outside the range seen in the apo states. Influences on binding-site variation could not be easily attributed to features such as ligand size or x-ray structure resolution. By combining these findings, we find that most binding site flexibility is compatible with the common practice in flexible docking, where backbones are kept rigid and side chains are allowed some degree of flexibility.

Prediction of disulfide dihedral angles using chemical shifts.

Cystine residues result from the formation of disulfide bonds between pairs of cysteine residues. This cross linking of the backbone is essential for the structure and activity of peptides and proteins. The conformation of a cystine side chain can be described using five dihedral angles, χ1, χ2, χ3, χ2', and χ1', with cystines favouring certain combinations of these angles. 2D NMR spectroscopy is ideally suited for structure determination of disulfide-rich peptides, because of their small size and constrained nature. However, only limited information of the cystine side chain conformation can be determined by NMR spectroscopy, leading to ambiguity in the deduced 3D structures. Resolving accurate structures is important as disulfide-rich peptides have proven to be promising drug candidates in a number of fields, either as bioactive leads or scaffolds. Using a database of NMR chemical shifts combined with crystallographic structures, we have developed a method called DISH that uses support vector machines to predict the dihedral angles of cysteine side chains. It is able to successfully predict χ2 angles with 91% accuracy, and has improved performance over existing prediction methods for χ1 angles, with 87% accuracy. For 81% of cysteine residues, DISH successfully predicted both the χ1 and χ2 angles. By revisiting published solution structures of peptides determined using NMR spectroscopy, we assessed the impact of additional cystine dihedral restraints on the quality of 3D models. DISH improved the resolution and accuracy, highlighting the potential for improving the understanding of structure-activity relationships and rational development of peptide drugs.

Privileged hydration sites in aromatic side chains: effect on conformational equilibrium.

Water interaction with peptide chains is one of the key structure stabilizing factors in an aqueous environment. Because of its strong polar character, water can bind to both anionic and cationic sites via electrostatic interactions. It can also act as a hydrogen-bond donor or acceptor according to its interactions with different polar groups in the backbone and side chains of peptides and proteins. Based on density functional theory calculations, the present report aims at illustrating the most energetically favorable interaction sites of aromatic side chains of phenylalanine, tyrosine, tryptophan, and histidine (neutral and protonated species) with surrounding water molecules. It was shown that beyond the strong interactions occurring between water and the aromatic ring acceptor/donor sites, such as O-H, N-H and -N[double bond, length as m-dash] groups, weaker interactions with π-electron clouds should also be considered. The latter type of binding, hereafter referred to as Hwπ interaction, involves one of the water hydrogen atoms (Hw) pointing toward the aromatic ring. Upon comparison between the theoretical data obtained from a purely implicit hydration model, i.e. a polarized solvent continuum, and those collected from a mixture of implicit and explicit hydration models, it has been shown that the explicit water molecule binding to aromatic rings affects the relative energies of the rotamers generated by the two side chain torsion angles (χ1 and χ2).

Conformational analysis of short polar side-chain amino-acids through umbrella sampling and DFT calculations.

Molecular and quantum mechanics calculations were carried out in a series of tripeptides (GXG, where X = D, N and C) as models of the unfolded states of proteins. The selected central amino acids, especially aspartic acid (D) and asparagine (N) are known to present significant average conformations in partially allowed areas of the Ramachandran plot, which have been suggested to be important in unfolded protein regions. In this report, we present the calculation of the propensity values through an umbrella sampling procedure in combination with the calculation of the NMR J-coupling constants obtained by a DFT model. The experimental NMR observations can be reasonably explained in terms of a conformational distribution where PPII and β basins sum up propensities above 0.9. The conformational analysis of the side chain dihedral angle (χ1), along with the computation of 3J(HαHβ), revealed a preference for the g - and g + rotamers. These may be connected with the presence of intermolecular H-bonding and carbonyl-carbonyl interactions sampled in the PPII and β basins. Taking into account all those results, it can be established that these residues show a similar behavior to other amino acids in short peptides regarding backbone φ,ψ dihedral angle distribution, in agreement with some experimental analysis of capped dipeptides.

Simulating the Function of the MjNhaP1 Transporter.

The structures of transport proteins have been steadily revealed in the last few decades, and yet the conversion of this information into molecular-level understanding of their function is still lagging behind. In this study, we try to elucidate how the action of the archaeal sodium/proton antiporter MjNhaP1 depends on its structure-energy relationship. To this end, we calculate the binding energies of its substrates and evaluate the conformational change barrier, focusing on the rotation of the catalytic residue D161. We find that sodium ions and protons compete against a common binding site and that the accessibility of this binding site is restricted to either the inside or outside of the cell. We suggest that the rotation of D161 χ1 angle correlates with the conformational change and is energetically unfavorable when D161 does not bind any substrate. This restriction ensures coupling between the sodium ions and the protons, allowing MjNhaP1 and probably other similar transporters to exchange substrates with minimal leak. Using Monte Carlo simulations we demonstrate the feasibility of our model. Overall we present a complete picture that reproduces the electroneutral (at 1:1 substrate ratio) and coupled transport activity of MjNhaP1 including the energetic basis for the criteria provided by Jardetzky half a century ago.

The Exact NOE as an Alternative in Ensemble Structure Determination

The structure-function paradigm is increasingly replaced by the structure-dynamics-function paradigm. All protein activity is steered by the interplay between enthalpy and entropy. Conformational dynamics serves as a proxy of conformational entropy. Therefore, it is essential to study not only the average conformation but also the spatial sampling of a protein on all timescales. To this purpose, we have established a protocol for determining multiple-state ensembles of proteins based on exact nuclear Overhauser effects (eNOEs). We have recently extended our previously reported eNOE data set for the protein GB3 by a very large set of backbone and side-chain residual dipolar couplings and three-bond J couplings. Here, we demonstrate that at least four structural states are required to represent the complete data set by dissecting the contributions to the CYANA target function, which quantifies restraint violations in structure calculation. We present a four-state ensemble of GB3, which largely preserves the characteristics obtained from eNOEs only. Due to the abundance of the input data, the ensemble and χ1 angles in particular are well suited for cross-validation of the input data and comparison to x-ray structures. Principal component analysis is used to automatically identify and validate relevant states of the ensembles. Overall, our findings suggest that eNOEs are a valuable alternative to traditional NMR probes in spatial elucidation of proteins.

Side Chain Conformational Distributions of a Small Protein Derived from Model-Free Analysis of a Large Set of Residual Dipolar Couplings.

Accurate quantitative measurement of structural dispersion in proteins remains a prime challenge to both X-ray crystallography and NMR spectroscopy. Here we use a model-free approach based on measurement of many residual dipolar couplings (RDCs) in differentially orienting aqueous liquid crystalline solutions to obtain the side chain χ1 distribution sampled by each residue in solution. Applied to the small well-ordered model protein GB3, our approach reveals that the RDC data are compatible with a single narrow distribution of side chain χ1 angles for only about 40% of the residues. For more than half of the residues, populations greater than 10% for a second rotamer are observed, and four residues require sampling of three rotameric states to fit the RDC data. In virtually all cases, sampled χ1 values are found to center closely around ideal g–, g+ and t rotameric angles, even though no rotamer restraint is used when deriving the sampled angles. The root-mean-square difference between experimental 3JHαHβ couplings and those predicted by the Haasnoot-parametrized, motion-adjusted Karplus equation reduces from 2.05 to 0.75 Hz when using the new rotamer analysis instead of the 1.1-Å X-ray structure as input for the dihedral angles. A comparison between observed and predicted 3JHαHβ values suggests that the root-mean-square amplitude of χ1 angle fluctuations within a given rotamer well is ca. 20°. The quantitatively defined side chain rotamer equilibria obtained from our study set new benchmarks for evaluating improved molecular dynamics force fields, and also will enable further development of quantitative relations between side chain chemical shift and structure.

Disulfide linkage Raman markers: a reconsideration attempt

During the last decades, Raman spectroscopy has been routinely used for probing the conformational features of disulfide linkages in peptides and proteins. However, the interpretation of disulfide Raman markers is currently performed by a simple rule derived from the earliest observations on dialkyl disulfides. More precisely, this rule consists of the following: (1) in analyzing the Raman bands in the 550–500 cm−1 region ascribed to disulfide bond stretch motion, namely, ν(S‐S), and (2) assigning the three types of Raman markers observed at ~500, ~520, and ~540 cm−1 to three families of rotamers defined along the three successive bonds of the ‐C‐S‐S‐C‐ moiety, referred to as ggg, ggt, and tgt. In this report, we attempt to show that an accurate analysis of disulfide vibrational features needs the use of the five torsion angles (χ1, χ2, χ3, χ2', and χ1') along the five successive bonds joining the two α‐carbon atoms in the cystine (Cys‐Cys) unit. The present work is inspired by the disulfide conformational investigations performed by a statistical scan of numerous protein crystal and nuclear magnetic resonance data, taking into account the handedness (right and left) of a disulfide bridge, its spatial shape (Staple, Hook, and Spiral), as well as the signs of the two extreme torsion angles χ1 and χ1'. It appears that the combined use of the old and recent conformational notations allows a more accurate structural and vibrational analysis of disulfide linkage. Copyright © 2014 John Wiley & Sons, Ltd.

Anomalies in the refinement of isoleucine.

A study of isoleucines in protein structures solved using X-ray crystallography revealed a series of systematic trends for the two side-chain torsion angles χ1 and χ2 dependent on the resolution, secondary structure and refinement software used. The average torsion angles for the nine rotamers were similar in high-resolution structures solved using either the REFMAC, CNS or PHENIX software. However, at low resolution these programs often refine towards somewhat different χ1 and χ2 values. Small systematic differences can be observed between refinement software that uses molecular dynamics-type energy terms (for example CNS) and software that does not use these terms (for example REFMAC). Detailing the standard torsion angles used in refinement software can improve the refinement of protein structures. The target values in the molecular dynamics-type energy functions can also be improved.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

The Power of Hard-Sphere Models: Explaining Side-Chain Dihedral Angle Distributions of Thr and Val

The energy functions used to predict protein structures typically include both molecular-mechanics and knowledge-based terms. In contrast, our approach is to develop robust physics- and geometry-based methods. Here, we investigate to what extent simple hard-sphere models can be used to predict side-chain conformations. The distributions of the side-chain dihedral angle χ1 of Val and Thr in proteins of known structure show distinctive features: Val side chains predominantly adopt χ1 = 180°, whereas Thr side chains typically adopt χ1 = 60° and 300° (i.e., χ1 = ±60° or g− and g+ configurations). Several hypotheses have been proposed to explain these differences, including interresidue steric clashes and hydrogen-bonding interactions. In contrast, we show that the observed side-chain dihedral angle distributions for both Val and Thr can be explained using only local steric interactions in a dipeptide mimetic. Our results emphasize the power of simple physical approaches and their importance for future advances in protein engineering and design.

Theoretical DFT karplus equations: Amino acid side‐chain torsion angle χ1

AbstractTheoretical Karplus equations for determining the conformation in proteins is applied to phenylalanine and tyrosine residues of Desulfovibrio vulgaris flavodoxin using density functional theory (DFT) calculations. The accuracy of different Karplus equations is evaluated showing that S2 Fourier coefficient is not negligible and C1 adopts positive values for some couplings. These two points should be considered in future empirical parameterizations. The dihedral angles χ1 calculated in this study using the full set of Fourier coefficients present a root mean square deviation of 6° against those obtained from X‐ray structures. © 2012 Wiley Periodicals, Inc.

Predicted structures of agonist and antagonist bound complexes of adenosine A3 receptor

We used the GEnSeMBLE Monte Carlo method to predict ensemble of the 20 best packings (helix rotations and tilts) based on the neutral total energy (E) from a vast number (10 trillion) of potential packings for each of the four subtypes of the adenosine G protein-coupled receptors (GPCRs), which are involved in many cytoprotective functions. We then used the DarwinDock Monte Carlo methods to predict the binding pose for the human A(3) adenosine receptor (hAA(3)R) for subtype selective agonists and antagonists. We found that all four A(3) agonists stabilize the 15th lowest conformation of apo-hAA(3)R while also binding strongly to the 1st and 3rd. In contrast the four A(3) antagonists stabilize the 2nd or 3rd lowest conformation. These results show that different ligands can stabilize different GPCR conformations, which will likely affect function, complicating the design of functionally unique ligands. Interestingly all agonists lead to a trans χ1 angle for W6.48 that experiments on other GPCRs associate with G-protein activation while all 20 apo-AA(3)R conformations have a W6.48 gauche+ χ1 angle associated experimentally with inactive GPCRs for other systems. Thus docking calculations have identified critical ligand-GPCR structures involved with activation. We found that the predicted binding site for selective agonist Cl-IB-MECA to the predicted structure of hAA(3)R shows favorable interactions to three subtype variable residues, I253(6.58), V169(EL2), and Q167(EL2), while the predicted structure for hAA(2A)R shows weakened to the corresponding amino acids: T256(6.58), E169(EL2), and L167(EL2), explaining the observed subtype selectivity.

Communication: Accurate determination of side-chain torsion angle χ1 in proteins: Phenylalanine residues

Quantitative side-chain torsion angle χ(1) determinations of phenylalanine residues in Desulfovibrio vulgaris flavodoxin are carried out using exclusively the correlation between the experimental vicinal coupling constants and theoretically determined Karplus equations. Karplus coefficients for nine vicinal coupling related with the torsion angle χ(1) were calculated using the B3LYP functional and basis sets of different size. Optimized χ(1) angles are in outstanding agreement with those previously reported by employing x ray and NMR measurements.

ChemInform Abstract: 7-Iodo-8-aza-7-deaza-2′-deoxyadenosine and 7-Bromo-8-aza-7-deaza-2′-deoxyadenosine.

The isomorphous structures of the title molecules, 4-amino-1-(2-deoxy-β-d-erythro-pento­furan­osyl)-3-iodo-1H-pyrazolo-[3,4-d]pyrimidine, (I), C10H12IN5O3, and 4-amino-3-bromo-1-(2-deoxy-β-d-erythro-pento­furan­osyl)-1H-pyrazolo[3,4-d]­pyrimidine, (II), C10H12BrN5O3, have been determined. The sugar puckering of both compounds is C1′-endo (1′E). The N-­glycosidic bond torsion angle χ1 is in the high-anti range [−73.2 (4)° for (I) and −74.1 (4)° for (II)] and the crystal structure is stabilized by hydrogen bonds.