Solvent Exchange Rates of Side-chain Amide Protons in Proteins

Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed.     

Temperature-dependence of protein hydrogen bond properties as studied by high-resolution NMR

The temperature-dependence of a large number of NMR parameters describing hydrogen bond properties in the protein ubiquitin was followed over a range from 5 to 65 degrees C. The parameters comprise hydrogen bond (H-bond) scalar couplings, h3JNC', chemical shifts, amide proton exchange rates, 15N relaxation parameters as well as covalent 1JNC' and 1JNH couplings. A global weakening of the h3JNC' coupling with increasing temperature is accompanied by a global upfield shift of the amide protons and a decrease of the sequential 1JNC' couplings. If interpreted as a linear increase of the N...O distance, the change in h3JNC' corresponds to an average linear thermal expansion coefficient for the NH-->O hydrogen bonds of 1.7 x 10(-4)/K, which is in good agreement with overall volume expansion coefficients observed for proteins. A residue-specific analysis reveals that not all hydrogen bonds are affected to the same extent by the thermal expansion. The end of beta-sheet beta1/beta5 at hydrogen bond E64-->Q2 appears as the most thermolabile, whereas the adjacent hydrogen bond I3-->L15 connecting beta-strands beta1 and beta2 is even stabilized slightly at higher temperatures. Additional evidence for the stabilization of the beta1/beta2 beta-hairpin at higher temperatures is found in reduced hydrogen exchange rates for strand end residue V17. This reduction corresponds to a stabilizing change in free energy of 9.7 kJ/mol for the beta1/beta2 hairpin. The result can be linked to the finding that the beta1/beta2 hairpin behaves as an autonomously folding unit in the A-state of ubiquitin under changed solvent conditions. For several amide groups the temperature-dependencies of the amide exchange rates and H-bond scalar couplings are uncorrelated. Therefore, amide exchange rates are not a sole function of the hydrogen bond "strength" as given by the electronic overlap of donors and acceptors, but are clearly dependent on other blocking mechanisms.     

A survey of hydrogen bond geometries in the crystal structures of amino acids

An analysis of the geometries of the hydrogen bonds observed by neutron diffraction in thirt-two crystal structures of amino acids shows the following results. Of the 168 hydrogen bonds in the data set, 64 involve the zwitterion groups 

and $\text{C}$O₂. Another 18 are from

to sulphate or carbonyl oxygens. The majority, 46, of these

H … O bonds are three-centered (bifurcated). Nine are four-centered (trifurcated). The geometry in which the three-centered hydrogen bond involves both oxygens of the same carboxylate group is not especially favoured. When it does occur, one hydrogen bond is generally shorter and the other longer, than when the bonding involves oxygens on different carboxylate groups. The shortest hydrogen bonds are the OH … O $\text{C}$, from a carboxylic acid hydroxyl to a carboxylate oxygen, and NH … O$\text{C}$ when the nitrogen is the ring atom in histidine or proline. Carboxylate groups, on average, accept six hydrogen bonds, with no examples of less than four bonds. The reason for the large number of three-centered

H … O$\text{C}$ bonds is therefore a proton deficiency arising from the disparity between the tripled donor property of the

groups and the sextuple, on average, acceptor property of the carboxylate groups. There is good geometrical evidence for the existence of H … O and H … Cl^? hydrogen bonds, especially involving the hydrogen atoms on α-atoms.     

NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors

The three-dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI-1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular beta-sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI-1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH-induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI-1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu-->Gln substitution at the P1' position in AMCI-1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI-1 structure.     

Evaluation of C-H cdots,three dots,centered O hydrogen bonds in native and misfolded proteins

Non-traditional C-H cdots, three dots, centered Y hydrogen bonds, in which a carbon atom acts as the hydrogen donor and an electronegative atom Y (Y=N, O or S) acts as the acceptor, have been reported in proteins, but their importance in protein structures is not well established. Here, we present the results of three computational tests that examine the significance of C-H cdots, three dots, centered Y bonds involving the C(alpha) in proteins. First, we compared the number of C(alpha)-H cdots, three dots, centered Y bonds in native structures with two sets of compact, energy-minimized decoy structures. The decoy structures contain about as many C(alpha)-H cdots, three dots, centered Y bonds as the native structures, indicating that the constraints of chain connectivity and compactness can lead to incidental formation of C(alpha)-H cdots, three dots, centered Y bonds. Secondly, we examined whether short C(alpha)-H cdots, three dots, centered Y bonds have a tendency to be linear, as is expected for a cohesive hydrogen-bonding interaction. The native structures do show this trend, but so does one of the decoy sets, suggesting that this criterion is also not sufficient to indicate a stabilizing interaction. Finally, we examined the preference for C(alpha)-H cdots, three dots, centered Y bond donors to be near to strong hydrogen bond acceptors. In the native proteins, the alpha protons attract strong acceptors like oxygen atoms more than weak acceptors. In contrast, hydrogen bond donors in the decoy structures do not distinguish between strong and weak acceptors. Thus, any individual C(alpha)-H cdots, three dots, centered Y bond may be fortuitous and occur due to the polypeptide connectivity and compactness. Taken collectively, however, C(alpha)-H cdots, three dots, centered Y bonds provide a weakly cohesive force that stabilizes proteins.     

A C-H triplebond O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins

The serendipitous observation of a C-H cdots, three dots, centered O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H triplebond O interaction between the T-4 C(alpha)H and T+1 Cz doublebond O group (C triplebond O< or =3.5A) becomes possible only when the T+1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T-4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T-4 position with the motif being further stabilized by the formation of a side-chain-backbone O triplebond H-N hydrogen bond involving the side-chain of residue T-4 and the N-H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H triplebond O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H cdots, three dots, centered O hydrogen bonds between (T-4) C(alpha)H triplebond O (T+1) and (T-8) C(alpha)H triplebondC doublebond O (T+3).     

Muon disrupts AKT hydrogen bond network in cancer

A cancer microenvironment generates strong hydrogen bond network system by the positive feedback loops supporting cancer complexity and robustness. Such network functions through the AKT locus generating high entropic energy supporting cancer metastatic robustness. Charged lepton particle muon follows the rule of Bragg effect during a collision with hydrogen network in cancer cells. Muon beam dismantles hydrogen bond network in cancer by the muon-catalyzed fusion, leading to apoptosis of cancer cells. Muon induces cumulative energy appearance on the hydrogen bond network in a cancer cell with its fast decay to an electron and two neutrinos. Thus, muon beam, muonic atom, muon neutrino shower, and electrons simultaneously cause fast neutralization of the AKT hydrogen bond network by the conversion of hydrogen into deuterium or helium, inactivating the hydrogen bond networks and inducing failure of cancer complexity and robustness with the disappearance of a malignant phenotype.     

Asparagine and glutamine differ in their propensities to form specific side chain-backbone hydrogen bonded motifs in proteins

Short range side chain‐backbone hydrogen bonded motifs involving Asn and Gln residues have been identified from a data set of 1370 protein crystal structures (resolution ≤ 1.5 Å). Hydrogen bonds involving residues i ? 5 to i + 5 have been considered. Out of 12,901 Asn residues, 3403 residues (26.4%) participate in such interactions, while out of 10,934 Gln residues, 1780 Gln residues (16.3%) are involved in these motifs. Hydrogen bonded ring sizes (C_n, where n is the number of atoms involved), directionality and internal torsion angles are used to classify motifs. The occurrence of the various motifs in the contexts of protein structure is illustrated. Distinct differences are established between the nature of motifs formed by Asn and Gln residues. For Asn, the most highly populated motifs are the C₁₀ (CO^δ_i …NH_{i + 2}), C₁₃ (CO^δ_i …NH_{i + 3}) and C₁₇ (N^δH_i …CO_{i ? 4}) structures. In contrast, Gln predominantly forms C₁₆ (CO^ε_i …NH_{i ? 3}), C₁₂ (N^εH_i …CO_{i ? 2}), C₁₅ (N^εH_i …CO_{i ? 3}) and C₁₈ (N^εH_i …CO_{i ? 4}) motifs, with only the C₁₈motif being analogous to the Asn C₁₇structure. Specific conformational types are established for the Asn containing motifs, which mimic backbone β‐turns and α‐turns. Histidine residues are shown to serve as a mimic for Asn residues in side chain‐backbone hydrogen bonded ring motifs. Illustrative examples from protein structures are considered. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

Counting peptide-water hydrogen bonds in unfolded proteins

It is often assumed that the peptide backbone forms a substantial number of additional hydrogen bonds when a protein unfolds. We challenge that assumption in this article. Early surveys of hydrogen bonding in proteins of known structure typically found that most, but not all, backbone polar groups are satisfied, either by intramolecular partners or by water. When the protein is folded, these groups form approximately two hydrogen bonds per peptide unit, one donor or acceptor for each carbonyl oxygen or amide hydrogen, respectively. But when unfolded, the backbone chain is often believed to form three hydrogen bonds per peptide unit, one partner for each oxygen lone pair or amide hydrogen. This assumption is based on the properties of small model compounds, like N-methylacetamide, or simply accepted as self-evident fact. If valid, a chain of N residues would have approximately 2N backbone hydrogen bonds when folded but 3N backbone hydrogen bonds when unfolded, a sufficient difference to overshadow any uncertainties involved in calculating these per-residue averages. Here, we use exhaustive conformational sampling to monitor the number of H-bonds in a statistically adequate population of blocked polyalanyl-six-mers as the solvent quality ranges from good to poor. Solvent quality is represented by a scalar parameter used to Boltzmann-weight the population energy. Recent experimental studies show that a repeating (Gly-Ser) polypeptide undergoes a denaturant-induced expansion accompanied by breaking intramolecular peptide H-bonds. Results from our simulations augment this experimental finding by showing that the number of H-bonds is approximately conserved during such expansion⇋compaction transitions.     

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.     

The ribbon of hydrogen bonds in globular proteins. IV. The example of the papain family

A study of the role of the hydrogen-bonding side chains in the ribbon of hydrogen bonds in globular proteins, using the papain family as an example, suggests that these side chains may be divided into three categories depending on their position in the molecule. In the first category, they form part of the local ribbon, in the second they form part of the ribbon at a site remote along the main chain, and in the third they play no role in the formation of the ribbon. The second case is particularly interesting because it provides a natural mechanism for the formation of the tertiary structure of the globular proteins. The results suggest that the robustness of the globular proteins towards mutations arises from the fact that many mutations that involve hydrogen-bonding side chains either leave the hydrogen bonding of the ribbon essentially unchanged or their hydrogen bonding plays no part in the formation of the ribbon in the first place. The results show that it is possible to obtain the ribbon of hydrogen bonds for a family of proteins whose data set's are of intermediate quality by studying the ribbons of several members of such a family and then taking an average over the different partial ribbons to create a standard ribbon of hydrogen bonds for the family as a whole. This method is used here to derive the standard ribbon for the papain family with papain itself, actinidin, and human liver cathepsin B as the representatives of the family. All three members of the family fit the standard ribbon with an accuracy of 85-91%. This result opens up the use of this technique for the study of a large number of globular proteins whose recorded data sets are of intermediate quality.     

A hydrogen bond study in tobacco mosaic virus using Moessbauer spectroscopy

The Moessbauer method was applied to obtain information on a suggested hydrogen bond in tobacco mosaic virus (TMV), between the hydroxyl group of Tyr 139 and a carboxyl oxygen of Glu 22 in a neighbouring subunit. Spectra of ¹²⁹I were taken of 3,5-di-iodo-l-tyrosine as a free amino acid and in situ in TMV. The increase of the pK value of 3,5-di-iodo-l-tyrosine by 0.8 units at position 139 in TMV compared to the free value is a strong argument in favour of the existence of a hydrogen bond via the relevant hydroxyl group.The reported study demonstrates the surprising sensitivity of the observable Moessbauer parameters to details of the electronic configuration in the neighbourhood of the probe nucleus.     

Quantum mechanical electronic structure calculation reveals orientation dependence of hydrogen bond energy in proteins

Hydrogen bond plays a unique role in governing macromolecular interactions with exquisite specificity. These interactions govern the fundamental biological processes like protein folding, enzymatic catalysis, molecular recognition. Despite extensive research work, till date there is no proper report available about the hydrogen bond's energy surface with respect to its geometric parameters, directly derived from proteins. Herein, we have deciphered the potential energy landscape of hydrogen bond directly from the macromolecular coordinates obtained from Protein Data Bank using quantum mechanical electronic structure calculations. The findings unravel the hydrogen bonding energies of proteins in parametric space. These data can be used to understand the energies of such directional interactions involved in biological molecules. Quantitative characterization has also been performed using Shannon entropic calculations for atoms participating in hydrogen bond. Collectively, our results constitute an improved way of understanding hydrogen bond energies in case of proteins and complement the knowledge‐based potential. Proteins 2017; 85:1046–1055. © 2017 Wiley Periodicals, Inc.     

Estimation of strength in different extra Watson-Crick hydrogen bonds in DNA double helices through quantum chemical studies

It was shown earlier, from database analysis, model building studies, and molecular dynamics simulations that formation of cross-strand bifurcated or Extra Watson-Crick hydrogen (EWC) bonds between successive base pairs may lead to extra rigidity to DNA double helices of certain sequences. The strengths of these hydrogen bonds are debatable, however, as they do not have standard linear geometry criterion. We have therefore carried out detailed ab initio quantum chemical studies using RHF/6-31G(2d,2p) and B3LYP/6-31G(2p,2d) basis sets to determine strengths of several bent hydrogen bonds with different donor and acceptors. Interaction energy calculations, corrected for the basis set superposition errors, suggest that N-H...O type bent EWC hydrogen bonds are possible along same strands or across the strands between successive base pairs, leading to significant stability (ca. 4-9 kcal/mol). The N-H...N and C-H...O type interactions, however, are not so stabilizing. Hence, consideration of EWC N-H...O H-bonds can lead to a better understanding of DNA sequence directed structural features.     

The effect of ring currents on carbon chemical shifts in cytochromes

Calculations suggest that some carbon chemical shifts in proteinsshould have large ring current shifts (>1 ppm). We present¹³C, ¹⁵N and ¹H assignments forcytochrome c₂ from Rhodospirillum rubrum, compare these withshifts for other cytochromes c, and show that the calculated ring currentshifts are similar to experimentally observed shifts, but that there remainsubstantial conformation-dependent shifts of side-chain carbons. Ringcurrent shifts as large as 6 ppm are observed. We show that the ring currenteffects do not seriously affect the Chemical Shift Index method fordelineating secondary structure, but may have an impact on more precisemethods for generating structural constraints.     

C alpha and C beta carbon-13 chemical shifts in proteins from an empirical database

We have constructed an extensive database of 13C C and C chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical - helix and -sheet geometries for C, and of ca. 2 ppm for C. The side-chain dihedral angle 1 has an effect of up to 0.5 ppm on the C shift, particularly for amino acids with branched side-chains at C. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different , shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and 1 angle, is 0.96 ppm for both C and C. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.     

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α‐helices compared to β‐sheets by ～0.2 kcal/mol. [2] Charge‐stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ～2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies.     

Vibrational coupling as diagnostic for intramolecular hydrogen bonds of carbohydrates in aqueous solution

The coupling of nuC-O and deltaO-D vibrations in the 1200-900 cm(-1) IR range leads to band shifting in opposite directions, which provides information on intramolecular hydrogen bonding of carbohydrates in aqueous solution. The aqueous solution IR spectra of 2-acetamide-1,6-anhydro-2-deoxy-D-glucopyranose and cis-1,2-cyclopentanediol and tetrahydrofuran-ethanol mixtures are reported. Frequency upshifting upon deuteration is observed for the nuC-O bands of both a hydrogen bond acceptor and donor in ether-hydroxyl and hydroxyl-hydroxyl contacts. The 1200-900 cm(-1) range can be used to reveal the presence of intramolecular hydrogen bonds in aqueous carbohydrates, unlike the OH stretching region, which cannot be used in this sense due to carbohydrate band masking by the strong nuOH IR absorption of water.     

Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers

Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding. In addition, we find that a pair of contacting TM helices is packed tighter when there are interhelical hydrogen bonds between them. We further describe several spatial motifs in the TM regions, including "Polar Clamp" and "Serine Zipper," where conserved Ser residues coincide with tightly packed locations in the TM region. With the examples of halorhodopsin, calcium-transporting ATPase, and bovine cytochrome c oxidase, we discuss the roles of hydrogen bonds in stabilizing helical bundles in polytopic membrane proteins and in protein functions.     

Nearest-neighbor effects on backbone alpha and beta carbon chemical shifts in proteins

We present a method for analyzing the chemical shift database to yield information on nearest-neighbor effects on carbon-13 chemical shift values for alpha and beta carbons of amino acids in proteins. For each amino acid sequence XYZ, we define two correction factors, Delta(XY) s and Delta(YZ) s , representing the effects on (delta13 Calpha-delta13 Cbeta) for residue Y from the preceding residue (X) and the following residue (Z), where X, Y, and Z represent one of the 20 naturally occurring amino acids, Delta designates the change in value or the correction factor (in ppm), and s is an index standing for one of three "pseudo secondary structure states" derived from chemical shift dispersions, which we show represent residues in primarily alpha-helix, beta-strand, and non-alphabeta(coil). The correction factors were obtained from maximum likelihood fitting of (delta13 Calpha-delta13 Cbeta) values from the chemical shifts of 651 proteins to a mixture of three Gaussians. These correction factors were derived strictly from the analysis of assigned chemical shifts, without regard to the three-dimensional structures of these proteins. The corrections factors were found to differ according to the secondary structural environment of the central residue (deduced from the chemical shift distribution) as well as by different identities of the nearest neighboring residues in the sequence. The areas subsumed by the sequence-dependent chemical shift distributions report on the relative energies of the sequences in different pseudo secondary structural environments, and the positions of the peaks indicate the chemical shifts of lowest energy conformations. As such, these results have potential applications to the determination of dihedral angle restraints from chemical shifts for structure determination and to more accurate predictions of chemical shifts in proteins of known structure. From a database of chemical shifts associated well-defined three-dimensional structures, comparisons were made between DSSP designations derived from three-dimensional structure and pseudo secondary structure designations derived from nearest-neighbor corrected chemical shift analysis. The high level of agreement between the two approaches to classifying secondary structure provides a measure of confidence in this chemical shift-based approach to the analysis of protein structure.