Intermolecular potentials for alpha-glycine from Raman and infrared scattering measurements

The frequencies of intermolecular modes in alpha-glycine-d0 and -d5 have been measured at 300 and 85 K by Raman and infrared scattering techniques. These frequencies were analyzed in terms of simple analytic interatomic potentials. Buckingham potentials were assumed for the nonbonded and hydrogen-bond interactions, and Coulomb and screened Coulomb potentials were assumed for the electrostatic interactions. The observed frequencies are well described by the simple model and the parameters of the hydrogen-bond potentials and the molecular charge distribution were determined from the analysis.     

Effective knowledge‐based potentials

Empirical or knowledge‐based potentials have many applications in structural biology such as the prediction of protein structure, protein–protein, and protein–ligand interactions and in the evaluation of stability for mutant proteins, the assessment of errors in experimentally solved structures, and the design of new proteins. Here, we describe a simple procedure to derive and use pairwise distance‐dependent potentials that rely on the definition of effective atomic interactions, which attempt to capture interactions that are more likely to be physically relevant. Based on a difficult benchmark test composed of proteins with different secondary structure composition and representing many different folds, we show that the use of effective atomic interactions significantly improves the performance of potentials at discriminating between native and near‐native conformations. We also found that, in agreement with previous reports, the potentials derived from the observed effective atomic interactions in native protein structures contain a larger amount of mutual information. A detailed analysis of the effective energy functions shows that atom connectivity effects, which mostly arise when deriving the potential by the incorporation of those indirect atomic interactions occurring beyond the first atomic shell, are clearly filtered out. The shape of the energy functions for direct atomic interactions representing hydrogen bonding and disulfide and salt bridges formation is almost unaffected when effective interactions are taken into account. On the contrary, the shape of the energy functions for indirect atom interactions (i.e., those describing the interaction between two atoms bound to a direct interacting pair) is clearly different when effective interactions are considered. Effective energy functions for indirect interacting atom pairs are not influenced by the shape or the energy minimum observed for the corresponding direct interacting atom pair. Our results suggest that the dependency between the signals in different energy functions is a key aspect that need to be addressed when empirical energy functions are derived and used, and also highlight the importance of additivity assumptions in the use of potential energy functions.     

Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.     

Argininamide binding arrests global motions in HIV-1 TAR RNA: comparison with Mg2+-induced conformational stabilization

The structure and dynamics of the stem-loop transactivation response element (TAR) RNA from the human immunodeficiency virus type-1 (HIV-1) bound to the ligand argininamide (ARG) has been characterized using a combination of a large number of residual dipolar couplings (RDCs) and trans-hydrogen bond NMR methodology. Binding of ARG to TAR changes the average inter-helical angle between the two stems from approximately 47 degrees in the free state to approximately 11 degrees in the bound state, and leads to the arrest of large amplitude (+/-46 degrees ) inter-helical motions observed previously in the free state. While the global structural dynamics of TAR-ARG is similar to that previously reported for TAR bound to Mg2+, there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogen-mediated 2hJ(N,N) scalar couplings as well as measured RDCs, the TAR-ARG complex is stabilized by a U38-A27.U23 base-triple involving an A27.U23 reverse Hoogsteen hydrogen bond alignment as well as by a A22-U40 Watson-Crick base-pair at the junction of stem I. These hydrogen bond alignments are not observed in either the free or Mg2+ bound forms of TAR. The combined conformational analysis of TAR under three states reveals that ligands and divalent ions can stabilize similar RNA global conformations through distinct interactions involving different hydrogen bond alignments in the RNA.     

Molecular Parameterisation and the Theoretical Calculation of Electrode Potentials

The difference in reduction potentials between ortho and para-benzoquinones has been calculated. The employs gas phase ab initio and semi-empirical computations in combination with free energy perturbation theory applied to gas and solution phase Monte Carlo simulations. The effects on calculated results of altering solute electrostatic parameterisation in solution phase simulations is examined. Atom centred charges derived from the molecular electrostatic potentials, MEPs, from optimised ab initio wavefunctions and charges generated by consideration of hydrogen bonded complexes are considered. Parameterisation of hydroxyl torsions in hydroquinone molecules is treated in a physically realistic manner. The coupled torsional system of the ortho-hydrobenzoquinone molecule is described by a potential energy surface calculated using gas phase AM1 semi-empirical computations rather than the simple torsional energy functions frequently employed in such calculations. Calculated differences in electrode potentials show that the electrostatic interactions of quinone and hydroquinone molecules in aqueous solution are not well described by atom centred charges derived from ab initio calculated MEPs. Moreover, results in good agreement with the experimental reduction potential difference can be obtained by employing high level ab initio calculations and solution phase electrostatic parameters developed by consideration of hydrogen bonded complexes.     

Simulating protein folding initiation sites using an alpha‐carbon‐only knowledge‐based force field

Protein folding is a hierarchical process where structure forms locally first, then globally. Some short sequence segments initiate folding through strong structural preferences that are independent of their three‐dimensional context in proteins. We have constructed a knowledge‐based force field in which the energy functions are conditional on local sequence patterns, as expressed in the hidden Markov model for local structure (HMMSTR). Carbon‐alpha force field (CALF) builds sequence specific statistical potentials based on database frequencies for α‐carbon virtual bond opening and dihedral angles, pair‐wise contacts and hydrogen bond donor‐acceptor pairs, and simulates folding via Brownian dynamics. We introduce hydrogen bond donor and acceptor potentials as α‐carbon probability fields that are conditional on the predicted local sequence. Constant temperature simulations were carried out using 27 peptides selected as putative folding initiation sites, each 12 residues in length, representing several different local structure motifs. Each 0.6 μs trajectory was clustered based on structure. Simulation convergence or representativeness was assessed by subdividing trajectories and comparing clusters. For 21 of the 27 sequences, the largest cluster made up more than half of the total trajectory. Of these 21 sequences, 14 had cluster centers that were at most 2.6 Å root mean square deviation (RMSD) from their native structure in the corresponding full‐length protein. To assess the adequacy of the energy function on nonlocal interactions, 11 full length native structures were relaxed using Brownian dynamics simulations. Equilibrated structures deviated from their native states but retained their overall topology and compactness. A simple potential that folds proteins locally and stabilizes proteins globally may enable a more realistic understanding of hierarchical folding pathways. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Structural hierarchy controls deformation behavior of collagen

The structure of collagen, the most abundant protein in mammals, consists of a triple helix composed of three helical polypeptide chains. The deformation behavior of collagen is governed by molecular mechanisms that involve the interaction between different helical hierarchies found in collagen. Here, we report results of Steered Molecular Dynamics study of the full-length collagen molecule (～290 nm). The collagen molecule is extended at various pulling rates ranging from 0.00003/ps to 0.012/ps. These simulations reveal a new level of hierarchy exhibited by collagen: helicity of the triple chain. This level of hierarchy is apparent at the 290 nm length and cannot be observed in the 7-9 nm models often described to evaluate collagen mechanics. The deformation mechanisms in collagen are governed by all three levels of hierarchy, helicity of single chain (level-1), helical triple helix (level-2), and hereby described helicity of the triple chain (level-3). The mechanics resulting from the three levels is described by an interlocking gear analogy. In addition, remarkably, the full-length collagen does not show much unwinding of triple helix unlike that exhibited by short collagen models. Further, the full-length collagen does not show significant unwinding of the triple helix, unlike that exhibited by short collagen. Also reported is that the interchain hydrogen bond energy in the full-length collagen is significantly smaller than the overall interchain nonbonded interaction energies, suggesting that the nonbonded interactions have far more important role than hydrogen bonds in the mechanics of collagen. However, hydrogen bonding is essential for the triple helical conformation of the collagen. Hence, although mechanics of collagen is controlled by nonbonded interchain interaction energies, the confirmation of collagen is attributed to the interchain hydrogen bonding.     

A computational method for design of connected catalytic networks in proteins

Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side‐chain functional groups which maximize transition‐state stabilization, and then searching for sites in protein scaffolds where the specified side‐chain–transition‐state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example, proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen‐bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw‐like two‐dimensional representation of the transition state with hydrogen‐bond donors, acceptors, and covalent interaction sites indicated, and all placements of side‐chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen‐bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully‐connected active sites in protein scaffolds. The method generates many fully‐connected active site solutions for a set of model reactions that are promising starting points for the design of fully‐preorganized enzyme catalysts.     

Determination of kinetic parameters from chemical relaxation data discrimination between coupled two-step binding reactions differing in stoichiometry

The chemical relaxation times of two different two-step equilibrium reactions, characterized by a 1:1 binding process followed by a subsequent rearrangement step and a stepwise 1:2 binding reaction, are analyzed for the purpose of qualitative model discrimination and quantitative determination of kinetic parameters. The equations describing the dependences of the two reciprocal relaxation times on suitable concentrations are given for both models in the general case as well as for four different limiting situations which are characterized by well separated relaxation times. The conditions corresponding to the limiting cases are expressed in terms of strong, weak and no coupling between the two partial equilibrium steps involved in both models. The coupling strength depends on the rate constants as well as on the total concentrations of the reactants. Criteria to discriminate between these two reaction models under defined limiting conditions are developed. In the general case, the product of both reciprocal relaxation times can be used to distinguish both models. If only one relaxation time can be resolved experimentally, it is possible under conditions described to determine only a reduced set of individual rate constants for most of the limiting cases considered. If both relaxation times are observed, all rate constants are determinable in the general case as well as in most of the limiting cases discussed.     

Kinase inhibitors and the case for CH...O hydrogen bonds in protein-ligand binding

Although the hydrogen bond is known to be an important mediator of intermolecular interactions, there has yet to be an analysis of the role of CH...O hydrogen bonds in protein-ligand complexes. In this work, we present evidence for such nonstandard hydrogen bonds from a survey of aromatic ligands in 184 kinase crystal structures and 358 high-resolution structures from the Protein Data Bank. CH groups adjacent to the positively charged nitrogen of nicotinamide exhibit geometric preferences strongly suggestive of hydrogen bonding interactions, as do heterocyclic CH groups in kinase ligands, while other aromatic CH groups do not exhibit these characteristics. Ab initio calculations reveal a considerable range of CH...O hydrogen bonding potentials among different aromatic ring systems, with nicotinamide and heterocycles preferred in kinase inhibitors showing particularly favorable interactions. These results provide compelling evidence for the existence of CH...O hydrogen bonds in protein-ligand interactions, as well as information on the relative strength of various aromatic CH donors. Such knowledge will be of considerable value in protein modeling, ligand design, and structure-activity analysis.     

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase.

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.     

Effect of hydrogen bond strength on the redox properties of phylloquinones: a two-dimensional hyperfine sublevel correlation spectroscopy study of photosystem I

The phylloquinones of photosystem I (PS I), A(1A) and A(1B), exist in near-equivalent protein environments but possess distinct thermodynamic and kinetic properties. Although the determinants responsible for the different properties of the phylloquinones are not completely understood, the strength and geometry of hydrogen bond interactions are significant factors in tuning and control of function. This study focuses on characterizing the hydrogen-bonding interactions of the phylloquinone acceptor, A(1A), by (1)H and (14)N HYSCORE spectroscopy. Photoaccumulation of PS I complexes at pH 8.0 results in the trapping of the phyllosemiquinone anion, A(1A)(-), on the A-branch of cofactors. The experiments described here indicate that A(1A)(-) forms a single H-bond. Using a simple point dipole approximation, we estimate its length to be 1.6 ± 0.1 ?. The value of the (1)H isotropic hyperfine coupling constant suggests that the H-bond has significant out-of-plane character. The (14)N HYSCORE spectroscopy experiments support the assignment of a H-bond wherein, the (14)N quadrupolar coupling constant is consistent with a backbone amide nitrogen as the hydrogen bond donor.     

Raman spectra and normal vibrations of dipeptides. I. Glycylglycine

We studied the Raman spectra of the zwitterionic glycylglycine crystal (GG) and its N-deuterated analog. A normal coordinate analysis on its α-crystalline form was performed and the effects of intra-and intermolecular couplings are discussed. A modified Urey-Bradley potential was used as a model of the intramolecular force field. Factor group splittings are described by the use of intermolecular potentials consisting of nonbonded atom-atom interactions and dipole-dipole interactions. Effects of hydrogen bonds on the vibrational frequencies of amino and carboxylate groups are also analyzed.     

Effect of intramolecular interactions on circular dichroism of ortho-substituted 1-phenethylamines

The synthesis of ortho-substituted (S)-1-phenethylamines via ortho-lithiation of its N,N-dimethyl derivative followed by reactions with different reagents is described. Circular dichroism spectra of synthesized compounds were measured. It is shown that the observed short-wave Cotton effects greatly depend on the existence of cyclic structure due to possible interactions such as hydrogen bond or electrostatic interaction.     

Energetics of water proton configurations in gas hydrates: comparison of various water models

The total interaction energies for a large number of water proton configurations in the unit cell of hydrate structure I consisting of 46 molecules are compared for qualitatively different water models, such as SPC/E, TIP4P, TIP5P, TIP 3f and AMOEBA. All calculations were carried out using the TINKER molecular modelling package. The Ewald summation method with metallic tin-foil boundary conditions is used to account for long-range electrostatic interactions. It was established that there is a high correlation between the energies calculated using the five water models (interaction potentials). The average correlation coefficient for all pairs of potentials is equal to 0.91. Analogous calculations were carried out to evaluate the consistency of the different water models with respect to a new property of the ice-like system: the hydrogen-bond-reversal asymmetry. It was established that, for all water models, there is relatively high correlation between the energy differences for proton configurations with opposite direction of all hydrogen bonds. In this case, the average correlation coefficient is 0.77. Data for the TIP4P potential differ noticeably from the others, especially owing to the variation in the total interaction energy. The validity and usefulness of simple discrete models of inter-molecular interactions are discussed.     

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).     

Conformation and packing analysis of polysaccharides and derivatives. I: Mannan

A previously described procedure for simultaneous optimization of bond lengths and angles was used to test different models for mannan I. Potential hydrogen bonds and the glycosidic angle were included in the optimization. A conformational model with bifurcated intramolecular hydrogen bonds of the type observed in the methyl cellobioside methanol complex showed the best agreement with available exprerimental data. The coordinates of this model were provided by computer calculations. The available X-ray data, however, were not sufficient for selecting this model; rather, ir data were necessary to furnish the needed information. The different conformational models tested all showed an almost constant virtual bond length O(1)–O(4) of the β-pyranose residue. This was in contrast to the previously obtained results for the α-pyranose residues.