Direct detection of N-H[...]N hydrogen bonds in biomolecules by NMR spectroscopy

A nuclear magnetic resonance (NMR) experiment is described for the direct detection of N-H[...]N hydrogen bonds (H-bonds) in 15N isotope-labeled biomolecules. This quantitative HNN-COSY (correlation spectroscopy) experiment detects and quantifies electron-mediated scalar couplings across the H-bond (H-bond scalar couplings), which connect magnetically active (15)N nuclei of the H-bond donor and acceptor. Detectable H-bonds comprise the imino H-bonds in canonical Watson-Crick base pairs, many H-bonds in unusual nucleic acid base pairs and H-bonds between protein backbone or side-chain N-H donor and N acceptor moieties. Unlike other NMR observables, which provide only indirect evidence of the presence of H-bonds, the H-bond scalar couplings identify all partners of the H-bond, the donor, the donor proton and the acceptor in a single experiment. The size of the scalar couplings can be related to H-bond geometries and as a time average to H-bond dynamics. The time required to detect the H-bonds is typically less than 1 d at millimolar concentrations for samples of molecular weight < or = approximately 25 kDa. A C15N/13C-labeled potato spindle tuber viroid T1 RNA domain is used as an example to illustrate this procedure.     

Mechanism of protein folding : III. Disulfide bonding

It was shown in lysozyme and phospholipase, and generally in proteins with disulfide bonds, that after the formation of secondary structures the hydrophobic interactions between the key pairs responsible for folding tertiary structures bring several cysteine residues close together. Among the possible combination of cysteine residues some definite pairs are realized in the tertiary structure. In the Appendix to this paper an algebraic relation is given which must be satisfied for two cysteine residues to make a disulficle bond. This relation is too strict to be applied to real problems, where the two cysteines come close together, but the distance is still too great to yield a disulfide bond. In this case the two residues can attract each other by disuifide formation potential. A geometrical graphic representation is given for the locus of the H atom of the SH group in the cysteine residue. This looks like a lampshade and provides us with a guide to select the correct choice among cysteine pairs. This method is applied to lysozyme and phospholipase to supplement the discussion of the preceding paper (T. Yoshimura, H. Noguchi, T. Inoue and N. Saitô, Biophys. Chem. 40 (1991) 277).     

Determinants of strand register in antiparallel beta-sheets of proteins.

Antiparallel beta-sheets present two distinct environments to inter-strand residue pairs: beta(A,HB) sites have two backbone hydrogen bonds; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only approximately 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic-Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi1 conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.     

Aldehyde dehydrogenase. Maintaining critical active site geometry at motif 8 in the class 3 enzyme.

Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue. Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335. These three residues were mutated conservatively. Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site.     

Aromatic side-chain interactions in proteins. I. Main structural features

In a data set of 593 nonhomologous proteins from the PDB, we have analyzed the pairing of phenylalanine, tyrosine, tryptophan, and histidine residues with their closest aromatic partner. The frequency distribution of the shortest interatomic distance of partners is bimodal with a sharp peak at approximately 3.8 A and a wider one at a longer distance. Only the 3.8 A peak corresponds to direct ring-ring interactions thus aromatic pairs. The aromatic pairs were separated into two classes, near-sequence pairs and far-sequence pairs. Near sequence pairs stabilize local structure, and far-sequence pairs stabilize tertiary structure. Far-sequence pairs (74% of all pairs) mainly bridge two beta-strands, followed by pairs that bridge a beta-strand and a helix, and pairs that bridge a beta-strand and a random coil structure. Pairs that bridge helices are rare. The secondary structure of the near-sequence pairs depends on the partner distance in the sequence. When the partners are 1, 3, or 4 residues apart in the sequence, pairs are mostly found in helical structures. When the partners are two apart, pairs are mostly found in the same beta-strand. Analysis of the frequency of near sequence pairs supports the hypothesis that aromatic pairing occurs after, rather than before, the formation of secondary structures.     

Hydrogen bonding interactions in noradrenaline-DMSO complexes: DFT and QTAIM studies of structure,properties and topology

The hydrogen bonding interactions between noradrenaline (NA) and DMSO were studied with density functional theory (DFT) regarding their geometries, energies, vibrational frequencies, and topological features of the electron density. The quantum theory of atoms in molecules (QTAIM) and the natural bond orbital (NBO) analyses were employed to elucidate the hydrogen bonding interaction characteristics in noradrenaline-DMSO complexes. The H-bonds involving the hydroxyls hydrogen in NA and the O atom in DMSO are dominant intermolecular H-bonds and are stronger than other H-bonds involving the methyl hydrogen of DMSO as a H-donor. The weak H-bonds also include a π H-bond which involves the benzene ring as a H-donor or H-acceptor. QTAIM identified the weak H-bonds formed between the methyl hydrogen of DMSO and the N atom in NA in some complexes (AB5, AB6 and AB7), which cannot be further confirmed by NBO and other methods, so there are probably no interactions between hydrogen and nitrogen atoms among these complexes. A good linear relationship between logarithmic electron density (lnρ _b ) at the bond critical point (BCP) and structural parameter (δR _H···Y) was found. The formations of new H-bonds in some complexes are helpful to strengthen the original intramolecular H-bond, this is attributed to the cooperativity of H-bonds in complexes and can be learned from the structure results and the NBO and QTAIM analyses. Analysis of various physically meaningful contributions arising from the energy decomposition procedures show that the orbital interactions of H-bond is predominant during the formation of the complex, moreover, both the hydrogen bonding interaction and the structural deformation are responsible for the stability of the complexes.     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.     

Intramolecular CH···O hydrogen bonds in the AI and BI DNA-like conformers of canonical nucleosides and their Watson-Crick pairs. Quantum chemical and AIM analysis

The aim of this work is to cast some light on the H-bonds in double-stranded DNA in its AI and BI forms. For this purpose, we have performed the MP2 and DFT quantum chemical calculations of the canonical nucleoside conformers, relative to the AI and BI DNA forms, and their Watson-Crick pairs, which were regarded as the simplest models of the double-stranded DNA. Based on the atoms-in-molecules analysis (AIM), five types of the CH···O hydrogen bonds, involving bases and sugar, were detected numerically from 1 to 3 per a conformer: C2'H···O5', C1'H···O2, C6H···O5', C8H···O5', and C6H···O4'. The energy values of H-bonds occupy the range of 2.3-5.6 kcal/mol, surely exceeding the kT value (0.62 kcal/mol). The nucleoside CH···O hydrogen bonds appeared to "survive" turns of bases against the sugar, sometimes in rather large ranges of the angle values, pertinent to certain conformations, which points out to the source of the DNA lability, necessary for the conformational adaptation in processes of its functioning. The calculation of the interactions in the dA·T nucleoside pair gives evidence, that additionally to the N6H···O4 and N1···N3H canonical H-bonds, between the bases adenine and thymine the third one (C2H···O2) is formed, which, though being rather weak (about 1 kcal/mol), satisfies the AIM criteria of H-bonding and may be classified as a true H-bond. The total energy of all the CH···O nontraditional intramolecular H-bonds in DNA nucleoside pairs appeared to be commensurable with the energy of H-bonds between the bases in Watson-Crick pairs, which implies their possible important role in the DNA shaping.     

Ribonuclease A. Analysis of the hydrogen bond geometry, and spatial accessibility at the active site

The hydrogen bonding of bovine ribonuclease A derived from the high resolution X-ray structure has been studied in detail. Correlations have been examined for main-chain-main-chain hydrogen bond angles, torsion angles and distances, respectively. Differences are found consistently for correlations associated with alpha-helix and beta-sheet, respectively. Ten of the 124 side-chains have four or more hydrogen bond contacts; two, including Glu-101, have five or more. Three potential C = O---H, three N---X and three potential side-chain H-bonds fail to form. A search for highly inaccessible buried residues resulted in nine outstanding examples, all of which are conserved across 38 known mammalian ribonuclease A sequences, indicating the importance of these residues for structural stability. Of the two histidines in the active site, His-12 has five hydrogen bonds and His-119 three. The conformational space accessible to these two catalytically important residues studied by means of simple non-bonded contact energy calculations confirms the existence of two alternative, interchangeable locations for His-119, while His-12 is locked in a local energy minimum.     

Characterizing the regularity of tetrahedral packing motifs in protein tertiary structure

MOTIVATION: While protein secondary structure is well understood, representing the repetitive nature of tertiary packing in proteins remains difficult. We have developed a construct called the relative packing group (RPG) that applies the clique concept from graph theory as a natural basis for defining the packing motifs in proteins. An RPG is defined as a clique of residues, where every member contacts all others as determined by the Delaunay tessellation. Geometrically similar RPGs define a regular element of tertiary structure or tertiary motif (TerMo). This intuitive construct provides a simple approach to characterize general repetitive elements of tertiary structure. RESULTS: A dataset of over 4 million tetrahedral RPGs was clustered using different criteria to characterize the various aspects of regular tertiary structure in TerMos. Grouping this data within the SCOP classification levels of Family, Superfamily, Fold, Class and PDB showed that similar packing is shared across different folds. Classification of RPGs based on residue sequence locality reveals topological preferences according to protein sizes and secondary structure. We find that larger proteins favor RPGs with three local residues packed against a non-local residue. Classifying by secondary structure, helices prefer mostly local residues, sheets favor at least two local residues, while turns and coil populate with more local residues. To depict these TerMos, we have developed 2 complementary and intuitive representations: (i) Dirichlet process mixture density estimation of the torsion angle distributions and (ii) kernel density estimation of the Cartesian coordinate distribution. The TerMo library and representations software are available upon request.     

Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme.

Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.     

Side chain dynamics and alternative hydrogen bonding in the mechanism of protein thermostabilization

To elucidate the mechanism of protein thermostabilization, the thermodynamic properties of small monomeric proteins from mesophilic and thermophilic organisms have been analyzed. Molecular dynamics simulations were employed in the study of dynamic features of charged and polar side chains of amino acid residues. The basic conclusion has been made: surface charged and polar side chains with high conformational mobility can form alternative hydrogen bonded (H-bonded) donor-acceptor pairs. The correlation between the quantitative content of alternative H-bonds per residue and the temperature of maximal thermostability of proteins has been found. The proposed mechanism of protein thermostabilization suggests continuous disruption of the primary H-bonds and formation of alternative ones, which maintain constant the enthalpy value in the native state and prevent a rapid increase of the conformational entropy with the rising temperature. The analysis of the results show that the more residues located in the N- and C-terminal regions and in the extended loops that are capable of forming alternative longer-range H-bonded pairs, the higher the protein thermostability.     

Amino acid code of protein secondary structure

The calculation of protein three-dimensional structure from the amino acid sequence is a fundamental problem to be solved. This paper presents principles of the code theory of protein secondary structure, and their consequence--the amino acid code of protein secondary structure. The doublet code model of protein secondary structure, developed earlier by the author (Shestopalov, 1990), is part of this theory. The theory basis are: 1) the name secondary structure is assigned to the conformation, stabilized only by the nearest (intraresidual) and middle-range (at a distance no more than that between residues i and i + 5) interactions; 2) the secondary structure consists of regular (alpha-helical and beta-structural) and irregular (coil) segments; 3) the alpha-helices, beta-strands and coil segments are encoded, respectively, by residue pairs (i, i + 4), (i, i + 2), (i, i = 1), according to the numbers of residues per period, 3.6, 2, 1; 4) all such pairs in the amino acid sequence are codons for elementary structural elements, or structurons; 5) the codons are divided into 21 types depending on their strength, i.e. their encoding capability; 6) overlappings of structurons of one and the same structure generate the longer segments of this structure; 7) overlapping of structurons of different structures is forbidden, and therefore selection of codons is required, the codon selection is hierarchic; 8) the code theory of protein secondary structure generates six variants of the amino acid code of protein secondary structure. There are two possible kinds of model construction based on the theory: the physical one using physical properties of amino acid residues, and the statistical one using results of statistical analysis of a great body of structural data. Some evident consequences of the theory are: a) the theory can be used for calculating the secondary structure from the amino acid sequence as a partial solution of the problem of calculation of protein three-dimensional structure from the amino acid sequence, and the calculated secondary structure and codon strength distribution can be used for simulating the next step of protein folding; b) one can propose that the same secondary structures can be folded into different tertiary structures and, vice versa, different secondary structures can be folded into the same tertiary structures, provided codon distributions are considered also; c) codons can be considered as first elements of protein three-dimensional structure language.     

Simulation of interactions between nucleic acid bases by refined atom-atom potential functions

Energy of interaction between nitrogen bases of nucleic acid has been calculated as a function of parameters determining the mutual position of two bases. Refined atom-atom potential functions are suggested. These functions contain terms proportional to the first (electrostatics), sixth (or tenth for the atoms forming a hydrogen bond) and twelfth (repulsion of all atoms) powers of interatomic distance. Calculations have shown that there are two groups of minima of the base interaction energy. The minima of the first group correspond to coplanar arrangement of the base pairs and hydrogen bond formation. The minima of the second group correspond to the position of bases one above the other in almost parallel planes. There are 28 energy minima corresponding to the formation of coplanar pairs with two (three for the G:C pair) almost linear N-H . . . O and (or) N-H . . . N hydrogen bonds. The position of nitrogen bases paired by two such H-bonds in any crystal of nucleic acid component in polynucleotide complexes and in tRNA is close to the position in one of these minima. Besides, for each pair there are energy minima corresponding to the formation of a single N-H . . . O or N-H . . . N and one C-H . . . O or C-H . . . N hydrogen bond. The form of potential surface in the vicinity of minima has been characterized. The results of calculations agree with the experimental data and with more rigorous calculations based on quantum-mechanical approach.     

Nucleobase pairing in expanded Watson-Crick-like genetic information systems

To guide the design of alternative genetic systems, we measured melting temperatures of DNA duplexes containing matched and mismatched nucleobase pairs from natural and unnatural structures. The pairs were analyzed in terms of structural features, including nucleobase size, number of hydrogen bonds formed, the presence of uncompensated hydrogen bonding functional groups, the nature of the bond joining the nucleobase to the sugar, and nucleobase charge. The results suggest that stability of nucleobase pairs correlates with the number of H-bonds, size complementarity, the presence of uncompensated functional groups, and the presence of charge on a nucleobase. Each of these properties appear to be more significant than the nature of the glycosidic bond and sequence context. The results provide guidelines for constructing stable Watson-Crick like nucleobase pairs with unnatural nucleobases. The experiments also demonstrate that expanded genetic systems can be constructed using size complementary nucleobase pairs that contain three hydrogen bonds.     

Long-term molecular dynamics simulation of copper plastocyanin in water

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.     

Modelling hydrogen bonds in NN-dimethylformamide

N, N-dimethylformamide (DMF) is a ‘universal’ solvent with the simplest amide structure. DMF has different interactions with many polymers and biomolecules. It is therefore necessary to study systematically the interactions in DMF itself first. In this study, both FT-IR and two molecular theoretical methods (MP2 and DFT/B3LYP) were used to study various hydrogen bonding interactions in DMF molecules based on its weak H-bonding donors CH/CH₃ and strong H-bonding acceptor C = O. The possible H-bonding donors and acceptors in DMF molecules were first analysed followed by modelling the effect of different structural environments on vC = O bands in infrared spectra. Finally, H-bonding properties including distance, angles and the energy as well as the probability of H-bonding patterns were obtained. The results showed that there exist five possible different weak types of H-bonding dimers; among them, three dimers consist of a pair of weak H-bonds, whereas two other dimers have two pairs of H-bonds, leading to 14 (including eight different) H-bonds. Two types of dimers were dominant, whereas three others can be omitted.     

Simulation of Interactions between Nucleic Acid Bases by Refined Atom-Atom Potential Functions

Abstract

Energy of interaction between nitrogen bases of nucleic acids has been calculated as a function of parameters determining the mutual position of two bases. Refined atom-atom potential functions are suggested. These functions contain terms proportional to the first (electrostatics), sixth (or tenth for the atoms forming a hydrogen bond) and twelfth (repulsion of all atoms) powers of interatomic distance. Calculations have shown that there are two groups of minima of the base interaction energy. The minima of the first group correspond to coplanar arrangement of the base pairs and hydrogen bond formation. The minima of the second group correspond to the position of bases one above the other in almost parallel planes. There are 28 energy minima corresponding to the formation of coplanar pairs with two (three for the G:C pair) almost linear N-H … O and (or) N-H … N hydrogen bonds. The position of nitrogen bases paired by two such H-bonds in any crystal of nucleic acid component, in polynucleotide complexes and in tRNA is close to the position in one of these minima. Besides, for each pair there are energy minima corresponding to the formation of a single N-H … O or N-H … N and one C-H … O or C-H … N hydrogen bond. The form of potential surface in the vicinity of minima has been characterized. The results of calculations agree with the experimental data and with more rigorous calculations based on quantum- mechanical approach.     

Study of the hydration characteristics of nucleic acid bases and their complementary pairs using the Monte-Carlo method

To elucidate the role of certain atomic groups in the formation of the nucleic acid hydrate shell, we simulated the systems involving a base or a complementary pair (the base molecules are methylated in N9 of purines and in N1 of pyrimidines) and 25 water molecules using the Monte-Carlo method. All hydrophilic centers, except for N1 purines and N3 pyrimidines in complementary pairs, form hydrogen bonds (H-bonds) with water molecules. The mean numbers of H-bonds formed by different centers, and distributions of the geometric characteristics of these bonds, which appeared similar to those in crystals, have been calculated. The formation of bridges of one, two of three water molecules between hydrophilic centers was shown. The probabilities of formation of these bridges have been calculated.