Bond Pattern Research Articles

An Ion-channel Modulator from the Saliva of the Brown Ear Tick has a Highly Modified Kunitz/BPTI Structure

Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 Å resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by β-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the ”Kunitz head” by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a β-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.

High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a 3 iron and Cu B of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH − groups do not fit, one water (or OH −) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu B ligand His326 is present in the imidazolate form.

Structure of Mixed Alkyllithium/Lithium Alkoxide Aggregates in Ethereal Solvents. Insights from Combined QM/MM Molecular Dynamics Simulations

Mixed alkyllithium/lithium alkoxides aggregates are important species in synthetic organic chemistry, but their electronic and geometric properties have not been extensively studied yet. The main objective of this work was to analyze the structure of simple prototypical aggregates in a coordinating solvent with the help of elaborated theoretical chemistry calculations. Within this aim, we have carried out molecular dynamics simulations for MeOLi, (EtLi)(MeOLi), and (EtLi)(2)(MeOLi)(2) systems in dimethyl ether solution. We use a combined QM/MM (quantum mechanics/molecular mechanics) force field that allows an appropriate description of the aggregate structure and of its interactions with the solvent. In the simulations, the aggregates are described at the B3LYP/6-31G(d) level while the solvent is described using the classical OPLS potential. For completeness, the influence of the chemical environment on the C-O-Li bond structure has been analyzed in some detail. The discussion focuses on (1) the distinctive properties of the alkoxide C-O-Li bond pattern, (2) the coordination of solvent molecules to the aggregates (number, stability), and (3) the time fluctuations of main structural parameters (Li-C and Li-O distances). We also show that nonclassical C-H...O hydrogen bonds involving H atoms of the solvent methyl groups and the O atom of the alkoxide are formed in the solvated MeOLi monomer and (EtLi)(MeOLi) dimer. Owing to these specific interactions, the monomer exhibits a nonlinear C-O-Li bond in solution.

A homogenized Love–Kirchhoff model for out-of-plane loaded random 2D lattices: Application to “quasi-periodic” brickwork panels

A homogenization procedure for finding the bending stiffness of a 2D regular lattice with random local interactions is proposed. The kinematic and static methods are used to provide explicit upper and lower bounds for the homogenized moduli. The proposed homogenization procedure is applied to a masonry obtained by a random perturbation of the periodic running bond masonry [Cecchi, A., Sab, K., 2009. Discrete and continuous models for in plane loaded random elastic brickwork. Eur. J. Mech. A 28, 610–625]. A numerical evaluation of the scatter between the discrete models and the 2D Love–Kirchhoff model is performed on a test case, for various values of the random perturbation parameter and of the parameter that characterizes the heterogeneity of the wall. As expected, when the number of heterogeneities in the structure is large enough, the average response of the random discrete model converges to an asymptotic response. It is shown that this asymptotic response is very close to that of the periodic discrete model which is in turn very close to the response of the deterministic homogenized model. Similarly to the conclusion of Cecchi and Sab [Cecchi A., Sab K., 2009. Discrete and continuous models for in plane loaded random elastic brickwork. Eur. J. Mech. A. 28, 610–625.] dedicated to in-plane loading, the present results concerning out-of-plane loading show (both by means of a discrete model and a homogenized model) that the running bond pattern may be used successfully to analyze historical masonries with blocks having irregular widths in the horizontal direction.

X-ray crystal structures and DFT calculations of differently charged aminocyclophosphazenes

Molecular structures of hexakis(amino)cyclotriphosphazene, N 3P 3(NH 2) 6, neutral and cationic forms of hexakis(ethylamino)cyclotriphosphazene, N 3P 3(NHC 2H 5) 6, and dicationic form of octakis(ethylamino)cyclotetraphosphazene, N 4P 4(NHC 2H 5) 8, were established through the X-ray diffraction experiments at 120(2), 165(2) and 110(2) K, respectively. Geometry of the six- and eight-membered cyclophosphazene rings of the molecules are discussed with the help of B3LYP/6-311G ∗∗ method/basis DFT calculations of their methylamino analogs and topological analysis of the electron density distribution. In all cases P–N bonds show high polar nature with considerable P → N charge transfer. Change of the P–N bond lengths upon the cycle protonation well correlated with the net charge redistribution at the P and N centers but separately for the endocyclic and exocyclic bonds. Presence of the additional π-bonding covalent contribution, as it is evidenced from the analysis of the bonds ellipticity, has to be suggested to explain these separate correlations. In the cyclotriphosphazene cation the finer details of the endocyclic P–N bond lengths changes can be attributed to the interplay of the variable π-bonding contribution with the change in the bond ionicity. In the crystal all compounds demonstrate large variety of bridged hydrogen bond patterns though the N–H…N and N–H…Cl interactions.

Docking and molecular dynamics simulation of quinone compounds with trypanocidal activity

In this work, two different docking programs were used, AutoDock and FlexX, which use different types of scoring functions and searching methods. The docking poses of all quinone compounds studied stayed in the same region in the trypanothione reductase. This region is a hydrophobic pocket near to Phe396, Pro398 and Leu399 amino acid residues. The compounds studied displays a higher affinity in trypanothione reductase (TR) than glutathione reductase (GR), since only two out of 28 quinone compounds presented more favorable docking energy in the site of human enzyme. The interaction of quinone compounds with the TR enzyme is in agreement with other studies, which showed different binding sites from the ones formed by cysteines 52 and 58. To verify the results obtained by docking, we carried out a molecular dynamics simulation with the compounds that presented the highest and lowest docking energies. The results showed that the root mean square deviation (RMSD) between the initial and final pose were very small. In addition, the hydrogen bond pattern was conserved along the simulation. In the parasite enzyme, the amino acid residues Leu399, Met400 and Lys402 are replaced in the human enzyme by Met406, Tyr407 and Ala409, respectively. In view of the fact that Leu399 is an amino acid of the Z site, this difference could be explored to design selective inhibitors of TR.

Water‐mediated Spectral Shifts in Rhodopsin and Bathorhodopsin†

The role of water molecules in spectral tuning of proteins has been left largely unexplored. This topic is important because changing hydrogen bond patterns during the activation process may lead to spectral shifts which can be of diagnostic value for the underlying structures. Arguments put forward in this article are based on spectral shift calculations of the rhodopsin and bathorhodopsin chromophore due to wat2a and 2b in the presence and absence of the counterion and of the amino acids lining the rhodopsin binding pocket. They show, among others, that a single water molecule can shift the absorbance by up to 0.1 eV or 34 nm depending on the environment of the chromophore.

Crystal Structures and Conformations of 5-Benzyl-2-thiohydantoin and Its 1-Acetylated Derivative

ABSTRACT The crystal structures of 5-benzyl-2-thiohydantoin (5-BTH) and 1-acetyl-5-benzyl-2-thiohydantoin (1-Ac-5-BTH) have been determined by X-ray diffraction. In the 5-BTH crystals, the enantiomeric (R)- and (S)-5-BTH molecules are connected to form cyclic dimers via the hydrogen bonds of the thioamide and the amide moieties. On the other hand, the intermolecular hydrogen bonds in 1-Ac-5-BTH crystals form an infinite chain. These differences in the hydrogen bond pattern are also discussed in the IR and Raman spectra. The ab initio molecular orbital calculations (Gaussian 03) with 6-31G(d,p) basis set were carried out for 5-BTH and 1-Ac-5-BTH to get the preferred conformation.

A Comparative Study into Two Dual Fluorescent Mechanisms via Positional Isomers of N-hydroxyarene-1,8-naphthalimides

Three isomers of hydroxy substituted N-aryl-1, 8-naphthalimides based on N-aryl naphthalic anhydride fluorophore have been synthesized. The decrease in fluorescence intensity from ortho to para substitution of hydroxy group on N-aryl reveals that para substituted isomer undergoes ESEC (Excited State with Extended Conjugation) mechanism which is proved by low quantum yield and appearance of dual emission. The ortho isomer, however, has high quantum yield and no tautomer emission, indicating ESIPT (Excited State Intramolecular Proton Transfer) mechanism is not operating. Similarly, all these isomers show strong fluorescence quenching in presence of strong H-bonding solvents like DMSO and pyridine, but there was neither the shift of emission bands nor the appearance of new bands for proton transfer to these solvents. Thus, it also indicates the absence of excited state proton transfer mechanism. Both the ortho isomer, and to a greater degree the meta isomer, showed larger quenching constants (Kapp) with pyridine than DMSO. This trend opposes the hydrogen-bond affinity for these solvents with phenol and points to a 2-point recognition interaction. In addition, a naphthalimide derivative using 2-aminoimidazole was prepared and examined for optimal positioning of a six-membered ring hydrogen bond pattern. No dual fluorescence was observed for this compound either.

A QM/MM Investigation of Thymine Dimer Radical Anion Splitting Catalyzed by DNA Photolyase

On the mend: The repair reaction of the thymine dimer by DNA photolyase (see picture) is studied by hybrid quantum mechanical/molecular mechanical dynamics simulations based on the X-ray structure of the enzyme-DNA complex. The dynamics of splitting of the thymine dimer radical anion within the DNA photolyase active site is characterized. The model includes the protein environment. DNA photolyase is a highly efficient light-driven enzyme that repairs the UV-induced cyclobutane pyrimidine dimer in damaged DNA. Herein, we investigate the repair reaction of the thymine dimer by means of hybrid quantum mechanical/molecular mechanical (QM/MM) dynamics simulations based on the X-ray structure of an enzyme-DNA complex. In analogy to the self-repair reaction, we find that the splitting mechanism of the cyclobutane ring is asynchronously concerted and is complete within a few picoseconds upon electron uptake. A few distinct processes characterize the dynamics of splitting of the thymine dimer radical anion within the DNA photolyase active site: continuous solvation reordering of the catalytic region, proton transfer from Glu283 to the dimer, as well as tight interactions of the cationic side chains of Arg232 and Arg350 with the thymine dimer. This points to the important role of the active-site hydrogen bond and salt-bridge patterns in stabilizing the thymine dimer anion and slowing down the electron back-transfer process. Comparison of the repair efficiency with respect to the self-repair reaction is also discussed.

MMH-2 as a new approach for the prediction of intermolecular interactions: the crystal packing of acetamide

A new approach (MMH-2) was applied and tested for the prediction of intermolecular interactions in the crystal packing of acetamide. In MMH-2, energies of random molecular interaction configurations are computed. It uses molecular association quantities from statistical thermodynamics in order to obtain intermolecular interaction motifs that follow a ranking process. The most important motifs are optimized. Here, the AM1 semiempirical Hamiltonian was applied for the calculation and optimization of each obtained configuration and a comparison to MP2 results is provided. Such a stepwise procedure follows the assumed genesis of crystal growth without using experimental input. For evaluation purposes, graph set analysis was used to classify the structural patterns of both acetamide polymorphs. It was also necessary to introduce a new geometrical similarity index for the comparison of calculated and experimental motifs. As a result, all experimental hydrogen bond patterns were found and molecular synthons in both polymorphic acetamide structures were predicted as local minima. This suggests a new strategy for crystal structure prediction of flexible molecules with a possible subsequent progress in crystal engineering in silico.

Role of Conserved Cysteines in the Alphavirus E3 Protein

Alphavirus particles are covered by 80 glycoprotein spikes that are essential for viral entry. Spikes consist of the E2 receptor binding protein and the E1 fusion protein. Spike assembly occurs in the endoplasmic reticulum, where E1 associates with pE2, a precursor containing E3 and E2 proteins. E3 is a small, cysteine-rich, extracellular glycoprotein that mediates proper folding of pE2 and its subsequent association with E1. In addition, cleavage of E3 from the assembled spike is required to make the virus particles efficiently fusion competent. We have found that the E3 protein in Sindbis virus contains one disulfide bond between residues Cys19 and Cys25. Replacing either of these two critical cysteines resulted in mutants with attenuated titers. Replacing both cysteines with either alanine or serine resulted in double mutants that were lethal. Insertion of additional cysteines based on E3 proteins from other alphaviruses resulted in either sequential or nested disulfide bond patterns. E3 sequences that formed sequential disulfides yielded virus with near-wild-type titers, while those that contained nested disulfide bonds had attenuated activity. Our data indicate that the role of the cysteine residues in E3 is not primarily structural. We hypothesize that E3 has an enzymatic or functional role in virus assembly, and these possibilities are further discussed.

Preferential binding specificity of silver cation to a single nucleobase over base pairs evaluated by abasic site-containing DNA

The binding specificity of silver cations to abasic (AP) site-containing DNA was electrochemically investigated by comparison with the fully matched DNA without the AP site. AP site-containing DNA is designed in a way that only the nucleotide opposite the AP site is variable to allow for coexistence of an unpaired nucleotide and a number of DNA base pairs. The surface of a gold electrode was modified by AP site-containing DNA duplex on which Ag + binding specificity was evaluated. Electrochemical investigations on the AP-DNA-modified electrodes reveal that Ag + preferentially associates to the unpaired nucleotides instead of the coexisted base pairs and shows sequence-dependant binding, especially stronger for purines than for pyrimidines. Additionally, the hydrogen bond pattern moieties of the unpaired nucleotides should be involved in Ag + binding evidenced by a decrease of the redox signal when introducing a ligand with its hydrogen bond moiety complementary to the nucleotide deoxycytidine. This is the first attempt to make a comparison in one DNA molecule for metal ion binding to coexisted unpaired nucleotide and DNA base pairs. The present method demonstrates an easy way for investigating binding specificity of heavy metal ions to AP site in the presence of coexisted DNA base pairs.

On the Importance of the Nature of Hydrogen Bond Donors in Multiple Hydrogen Bond Systems

AbstractAssociation constants for the reaction between a fourfold hydrogen bond receptor 4 possessing an AADA hydrogen bond pattern and several complementary partners 5–7 with DDAD and DDA patterns have been determined by NMR titration and by isothermal titration calorimetry (ITC). A variation of one donor site in the DDAD pattern resulted in the following, surprising sequence of stability of the complexes: KAss(6, X = NHCOR) > KAss(7, X = H) > KAss(5, X = NHR). The synthesis of a well soluble DDAD receptor, the amidopyridylurea 6b with a triethylene glycol chain, is described. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

E9-Im9 colicin DNase-immunity protein biomolecular association in water: a multiple-copy and accelerated molecular dynamics simulation study.

Protein−protein transient and dynamic interactions underlie all biological processes. The molecular dynamics (MD) of the E9 colicin DNase protein, its Im9 inhibitor protein, and their E9-Im9 recognition complex are investigated by combining multiple-copy (MC) MD and accelerated MD (aMD) explicit-solvent simulation approaches, after validation with crystalline-phase and solution experiments. Im9 shows higher flexibility than its E9 counterpart. Im9 displays a significant reduction of backbone flexibility and a remarkable increase in motional correlation upon E9 association. Im9 loops 23−31 and 54−64 open with respect to the E9-Im9 X-ray structure and show high conformational diversity. Upon association a large fraction (∼20 nm2) of E9 and Im9 protein surfaces become inaccessible to water. Numerous salt bridges transiently occurring throughout our six 50 ns long MC-MD simulations are not present in the X-ray model. Among these Im9 Glu31−E9 Arg96 and Im9 Glu41−Lys89 involve interface interactions. Through the use of 10 ns of Im9 aMD simulation, we reconcile the largest thermodynamic impact measured for Asp51Ala mutation with Im9 structure and dynamics. Lys57 acts as an essential molecular switch to shift Im9 surface loop towards an ideal configuration for E9 inhibition. This is achieved by switching Asp60−Lys57 and Asp62−Lys57 hydrogen bonds to Asp51−Lys57 salt bridge. E9-Im9 recognition involves shifts of conformational distributions, reorganization of intramolecular hydrogen bond patterns, and formation of new inter- and intramolecular interactions. The description of key transient biological interactions can be significantly enriched by the dynamic and atomic-level information provided by computer simulations.

Characterization of Chitin and Chitosan Molecular Structure in Aqueous Solution.

Molecular dynamics simulations have been used to characterize the structure of single chitin and chitosan chains in aqueous solutions. Chitin chains, whether isolated or in the form of a β-chitin nanoparticle, adopt the 2-fold helix with ϕ and φ values similar to its crystalline state. In solution, the intramolecular hydrogen bond HO3(n)···O5(n+1) responsible for the 2-fold helical motif in these polysaccharides is stabilized by hydrogen bonds with water molecules in a well-defined orientation. On the other hand, chitosan can adopt five distinct helical motifs, and its conformational equilibrium is highly dependent on pH. The hydrogen bond pattern and solvation around the O3 atom of insoluble chitosan (basic pH) are nearly identical to these quantities in chitin. Our findings suggest that the solubility and conformation of these polysaccharides are related to the stability of the intrachain HO3(n)···O5(n+1) hydrogen bond, which is affected by the water exchange around the O3-HO3 hydroxyl group.

Analysis of structure and function of the giant protein Pf332 in Plasmodium falciparum

Virulence of Plasmodium falciparum, the most lethal parasitic disease in humans, results in part from adhesiveness and increased rigidity of infected erythrocytes. Pf332 is trafficked to the parasite-infected erythrocyte via Maurer's clefts, structures for protein sorting and export in the host erythrocyte. This protein has a domain similar to the Duffy-binding-like (DBL) domain, which functions by binding to receptors for adherence and invasion. To address structure of the Pf332 DBL domain, we expressed this region, and validated its fold on the basis of the disulphide bond pattern, which conformed to the generic pattern for DBL domains. The modelled structure for Pf332 DBL had differences compared with the erythrocyte-binding region of the αDBL domain of Plasmodium knowlesi Duffy-binding protein (Pkα-DBL). We addressed the function of Pf332 by constructing parasites that either lack expression of the protein or express an altered form. We found no evidence that Pf332 is involved in cytoadhesion or merozoite invasion. Truncation of Pf332 had a significant effect on deformability of the P. falciparum-infected erythrocyte, while loss of the full protein deletion did not. Our data suggest that Pf332 may contribute to the overall deformability of the P. falciparum-infected erythrocyte by anchoring and scaffolding.

The Catalytic Activity of Protein-disulfide Isomerase Requires a Conformationally Flexible Molecule

Protein-disulfide isomerase (PDI) catalyzes the formation of the correct pattern of disulfide bonds in secretory proteins. A low resolution crystal structure of yeast PDI described here reveals large scale conformational changes compared with the initially reported structure, indicating that PDI is a highly flexible molecule with its catalytic domains, a and a', representing two mobile arms connected to a more rigid core composed of the b and b' domains. Limited proteolysis revealed that the linker between the a domain and the core is more susceptible to degradation than that connecting the a' domain to the core. By restricting the two arms with inter-domain disulfide bonds, the molecular flexibility of PDI, especially that of its a domain, was demonstrated to be essential for the enzymatic activity in vitro and in vivo. The crystal structure also featured a PDI dimer, and a propensity to dimerize in solution and in the ER was confirmed by cross-linking experiments and the split green fluorescent protein system. Although sedimentation studies suggested that the self-association of PDI is weak, we hypothesize that PDI exists as an interconvertible mixture of monomers and dimers in the endoplasmic reticulum due to its high abundance in this compartment.

Discrete and continuous models for in plane loaded random elastic brickwork

Abstract The aim of this paper is to study non-periodic masonries – typical of historical buildings – by means of a perturbation approach and to evaluate the effect of a random perturbation on the elastic response of a periodic masonry wall. The random masonry is obtained starting from a periodic running bond pattern. A random perturbation on the horizontal positions of the vertical interfaces between the blocks which form the masonry wall is introduced. In this way, the height of the blocks is uniform, while their width in the horizontal direction is random. The perturbation is limited such as each block has still exactly 6 neighboring blocks. In a first discrete model, the blocks are modeled as rigid bodies connected by elastic interfaces (mortar thin joints). In other words, masonry is seen as a “skeleton” in which the interactions between the rigid blocks are represented by forces and moments which depend on their relative displacements and rotations. A second continuous model is based on the homogenization of the discrete model. Explicit upper and lower bounds on the effective elastic moduli of the homogenized continuous model are obtained and compared to the well-known effective elastic moduli of the regular periodic masonry. It is found that the effective moduli are not very sensitive to the random perturbation (less than 10%). At the end, the Monte Carlo simulation method is used to compare the discrete random model and the continuous model at the structural level (a panel undergoing in plane actions). The randomness of the geometry requires the generation of several samples of size L of the discrete masonry. For a sample of size L , the structural discrete problem is solved using the same numerical procedure adopted in [Cecchi, A., Sab, K., 2004. A comparison between a 3D discrete model and two homogenized plate models for periodic elastic brickwork, International Journal of Solids Structures 41 (9–10), 2259–2276] and the average solution over the samples gives an estimation which depends on L . As L increases, an asymptotic limit is reached. One issue is to find the minimum size for L and to compare the asymptotic average solution to the one obtained from the continuous homogenized model.

The NMR Structures of the Major Intermediates of the Two-domain Tick Carboxypeptidase Inhibitor Reveal Symmetry in Its Folding and Unfolding Pathways

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.