Formation Of Hydrogen Bonding Interactions Research Articles

Crystal Structure of a Heat-resilient Phytase from Aspergillus fumigatus, Carrying a Phosphorylated Histidine

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 Å resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A. fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A. niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A. fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.

Crystal Structure of a Heat-resilient Phytase from Aspergillus fumigatus, Carrying a Phosphorylated Histidine

In order to understand the structural basis for the high thermostability of phytase from Aspergillus fumigatus, its crystal structure was determined at 1.5 Å resolution. The overall fold resembles the structure of other phytase enzymes. Aspergillus niger phytase shares 66% sequence identity, however, it is much less heat-resistant. A superimposition of these two structures reveals some significant differences. In particular, substitutions with polar residues appear to remove repulsive ion pair interactions and instead form hydrogen bond interactions, which stabilize the enzyme; the formation of a C-terminal helical capping, induced by arginine residue substitutions also appears to be critical for the enzyme's ability to refold to its active form after denaturation at high temperature. The heat-resilient property of A. fumigatus phytase could be due to the improved stability of regions that are critical for the refolding of the protein; and a heat-resistant A. niger phytase may be achieved by mutating certain critical residues with the equivalent residues in A. fumigatus phytase. Six predicted N-glycosylation sites were observed to be glycosylated from the experimental electron density. Furthermore, the enzyme's catalytic residue His59 was found to be partly phosphorylated and thus showed a reaction intermediate, providing structural insight, which may help understand the catalytic mechanism of the acid phosphatase family. The trap of this catalytic intermediate confirms the two-step catalytic mechanism of the acid histidine phosphatase family.

Changes in drug 13C NMR chemical shifts as a tool for monitoring interactions with DNA

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)] 2 to monitor drug 13C NMR chemical shifts changes. The binding mode of netropsin to the minor groove of DNA is well-known, and served as a good model for evaluating the relative sensitivity of 13C chemical shifts to hydrogen bonding. Large downfield shifts were observed for four resonances of carbons that neighbor sites which are known to form hydrogen bond interactions with the DNA minor groove. Many of the remaining resonances of netropsin exhibit shielding or relatively smaller deshielding changes. Based on the model system presented here, large deshielding NMR shift changes of a ligand upon macromolecule binding can likely be attributed to hydrogen bond formation at nearby sites.

Alanine-scanning mutagenesis of transmembrane domain 6 of the M(1) muscarinic acetylcholine receptor suggests that Tyr381 plays key roles in receptor function.

Transmembrane domain 6 of the muscarinic acetylcholine (ACh) receptors is important in ligand binding and in the conformational transitions of the receptor but the roles of individual residues are poorly understood. We have carried out a systematic alanine-scanning mutagenesis study on residues Tyr381 to Val387 within the binding domain of the M(1) muscarinic ACh receptor. The seven mutations were then analyzed to define the effects on receptor expression, agonist and antagonist binding, and signaling efficacy. Tyr381Ala produced a 40-fold reduction in ACh affinity and a 50-fold reduction in ACh-signaling efficacy. Leu386Ala had similar but smaller effects. Asn382Ala caused the largest inhibition of antagonist binding. The roles of the hydroxyl group and benzene ring of Tyr381 were probed further by comparative analysis of the Tyr381Phe and Tyr381Ala mutants using three series of ligands: ACh analogs, azanorbornane- and quinuclidine-based ligands, and atropine analogs. These data suggested that the hydroxyl group of Tyr381 is primarily involved in forming hydrogen bond interactions with the oxygen atoms present in the side chain of ACh. We propose that this interaction is established in the ground state and preserved in the activated state of the receptor. In contrast, the Tyr381 benzene ring may form a cation-pi interaction with the positively charged head group of ACh that contributes to the activated state of the receptor but not the ground state. However, the hydroxyl group and benzene ring of Tyr381 both participate in interactions with azanorbornane- and quinuclidine-based ligands and atropine analogs in the ground state as well as the activated state of the receptor.

Structural model of human glucokinase in complex with glucose and ATP: implications for the mutants that cause hypo- and hyperglycemia.

Mutations in human glucokinase are implicated in the development of diabetes and hypoglycemia. Human glucokinase shares 54% identical amino acid residues with human brain hexokinase I. This similarity was used to model the structure of glucokinase by analogy to the crystal structure of brain hexokinase. Glucokinase was modeled with both its substrates, glucose and MgATP, to understand the effect of mutations. The glucose is predicted to form hydrogen bond interactions with the side chains of glucokinase residues Thr 168, Lys 169, Asn 204, Asp 205, Asn 231, and Glu 290, similar to those observed for brain hexokinase I. The magnesium ion is coordinated by the carboxylates of Asp 78 and Asp 205 and the gamma-phosphate of ATP. ATP is predicted to form hydrogen bond interactions with residues Gly 81, Thr 82, Asn 83, Arg 85, Lys 169, Thr 228, Lys 296, Thr 332, and Ser 336. Mutations of residues close to the predicted ATP binding site produced dramatic changes in the Km for ATP, the catalytic rate, and a loss of cooperativity, which confirmed our model. Mutations of residues in the glucose binding site dramatically reduced the catalytic activity, as did a mutation that was predicted to disrupt an alpha-helix. Other mutations located far from the active site gave smaller changes in kinetic parameters. In the absence of a crystal structure for glucokinase, our models help rationalize the potential effects of mutations in diabetes and hypoglycemia, and the models may also facilitate the discovery of pharmacological glucokinase activators and inhibitors.

Synthesis and crystal structures of 4,4'-bipyridinium and 2,2'-bipyridinium pentachloro complexes containing the polynuclear anions [Bi2Cl10]4- and [Bi4Cl20]8-

The compounds 4,4′-bipyridinium(2+) pentachloro-bismuthate(III) (1), [4,4′-(C10H8N2)BiCl5] and 2,2′-bipyridinium(2+) pentachloro-bismuthate(III) (2), [2,2′-(C10H8N2)Bi2Cl10] have been obtained by reacting bismuthate oxide and 4,4′-bipyridine or 2,2′-bipyridine in HCl acid medium. They have been characterized by single crystal X-ray analysis. (1) crystallizes in the triclinic space group $$P\bar 1$$ with a = 9.776(2), b = 11.009(3),c = 8.346(1) A, α = 101.58(2), β = 98.63(2), and γ = 112.86(2)°. (2) crystallizes in the monoclinic space group P21/c with a = 14.239(2), b = 14.226(2), c = 16.275(3) A, and β = 110.15(2)°. The crystal structure of (1) consists of 4,4′-bipyridinium(2+) cations interacting through hydrogen bonding with [Bi2Cl10]2− dimers giving rise to endless double chains, while that of (2) is formed by 2,2′-bipyridinium(2+) cations and [Bi4Cl20]8− tetramers extensively interacting through hydrogen bonding. The different polynuclearity of the anions seems related to the different directions along which each cation can form hydrogen bond interactions.

Investigation on coordination ability of N-chloro-acetylglycine towards Cu(II) in solid and solution state

The solution and solid state behavior of the binary system Cu(II)- N-chloroacetylglycine (Cl-acglyH) and the corresponding ternary systems with 2,2′-bipyridine (bpy) and 1,10-phenanthroline (ophen) were investigated by means of pH-metric and spectrophotometric titrations. The X-ray crystal structure of the complex [Cu(ophen)(Cl-acgly) 2]·2H 2O (Cl-acgly = N-chloroacetylglycine monoanion) is also reported. The crystals of the compound C 20Cl 2CuN 4O 8H 22 are monoclinic, space group P2 1/ c, a = 17.574(3), b = 7.125(2), c = 25.113(6) Å, β = 130.04(2)°, Z = 4, R = 0.077, R w = 0.088. The structure consists of monomeric [Cu(ophen)(Clgly) 2] units and lattice water molecules. The Cu(II) atom is square planar coordinated by two carboxylic oxygens of two amino acid molecules and two nitrogen atoms of the ophen molecule. Two long contacts involve the uncoordinated carboxylic oxygens. Crystal packing is due to ring-stacking interaction involving ophen molecules and to a strong intramolecular hydrogen bond involving the chlorine atom and the amidic nitrogen of one ligand molecule. In solution the species [CuL 2] and [CuALOH] (A = bpy or ophen) prevail in the binary and ternary system, respectively, both arising from carboxylic-oxygen-metal coordination of the amino acid molecule. The coordination of the OH − group in the ternary species prevents metal hydrolysis up to pH 11. The possibility of forming hydrogen bond interactions involving the chlorine atom seems the to be determinant factor in stabilizing the ternary complexes.

Models of HIV-1 protease with peptides representing its natural substrates

The substrate specificity of HIV-1 protease has been investigated by molecular modeling of HIV-1 protease with seven peptides representing the naturally occurring cleavage sites in the gag and gag–pol polyprotein precursors. These peptides contain a wide range of amino acids, although only hydrophobic residues are found at either side of the scissile peptide bond. Polar amino acid side chains in each substrate may form hydrogen bond interactions with atoms of HIV protease. HIV-1 protease residues Arg 8, Asp 29, Asp 30, Lys 45, Met 46, Gly 49 and Pro 81 were predicted to form hydrogen bond interactions with polar substrate side chains that may provide sequence specific recognition of the substrates. Several of these protease residues are mutated in inhibitor resistant strains of HIV. Residues equivalent to HIV-1 protease Asp 30 and Pro 81 have been shown to be critical components for substrate recognition. The calculated interaction energy betwen HIV protease and the tetrahedral intermediate of the substrates has a correlation coefficient of 71% with the differences in free energy calculated from kinetic measurements. The implications for the reaction pathway are discussed.

Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions.

Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser 138 as a member of a catalytically important "triad" of residues also involving Tyr151 and Lys155. A Ser-->Ala exchange at position 138 results in an almost complete (> 99.9%) loss of enzymatic activity, which is not observed with a Ser-->Thr replacement. This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands beta A, beta D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme and with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3 alpha/20 beta-HSD from Streptomyces hydrogenans suggests a hydrogen bond in that enzyme between the Thr12 side chain and the backbone NH of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the beta D strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of kcat/Km in the dehydrogenase direction but also unchanged 3-oxoreductase properties. It appears that the binding of NAD+ to the protein is influenced by local structural changes involving strand beta D and turn beta A to alpha B.

Effects of molecular structure upon complex‐support interactions

AbstractTwo cationic, copper metal complexes with different ligands were synthesized: ethylenediamine (en) [Cu(C2N8N2) (ClO4)2] and diethanolamine (Deta) {[Cu‐C8H21N2O4]ClO4}. These complexes were mounted on Cab‐O‐Sil using nonaqueous impregnation techniques, and the loadings were determined at which multilayers formed. These samples were analyzed for copper, carbon, nitrogen and hydrogen content to determine if ligand dissociation occurred during the impregnation. Samples were decomposed in a thermal gravimetric apparatus to determine the kinetics of the thermolysis reaction in air, and the evolved gases were analyzed by gas chromatograph‐mass spectrometry to determine the products of the thermolysis reaction. The results of these and earlier studies are summarized in a model that describes the effects of molecular structure upon complex‐support interactions. Complexes with the ability to form hydrogen bond interactions between the ligand and the support form strong interactions with the surface of silica, whereas complexes without such hydrogen bond interactions are only weakly attracted to the silica surface. Strong interactions with the surface may also arise as a result of ligand dissociation and direct interaction of the metal ion with the surface oxygens. The charge on the complex and its shape play less important roles in determining the affinity of the metal complex with the silica.

Molecular structure of flavocytochrome b2 at 24 Å resolution

The crystal structure of flavocytochrome b 2 has been solved at 3.0 Å resolution by the method of multiple isomorphous replacement with anomalous scattering. Area detector data from native and two heavy-atom derivative crystals were used. The phases were refined by the B. C. Wang phase-filtering procedure utilizing the 67% ( v v ) solvent content of the crystals. A molecular model was built first on a minimap and then on computer graphics from a combination of maps both averaged and not averaged about the molecular symmetry axis. The structure was extended to 2.4 Å resolution using film data recorded at a synchrotron and refined by the Hendrickson-Konnert procedure. The molecule, a tetramer of M r 230,000, is located on a crystallographic 2-fold axis and possesses local 4-fold symmetry. Each subunit is composed of two domains, one binding a heme and the other an FMN prosthetic group. In subunit 1, both the cytochrome and the flavin-binding domain are visible in the electron density map. In subunit 2 the cytochrome domain is disordered. However, in the latter, a molecule of pyruvate, the product of the enzymatic reaction, is bound at the active site. The cytochrome domain consists of residues 1 to 99 and is folded in a fashion similar to the homologous soluble fragment of cytochrome b 5. The flavin binding domain contains a parallel β 8 α 8 barrel structure and is composed of residues 100 to 486. The remaining 25 residues form a tail that wraps around the molecular 4-fold axis and is in contact with each remaining subunit. The FMN moiety, which is located at the C-terminal end of the central β-barrel, is mostly sequestered from solvent; it forms hydrogen bond interactions with main- and side-chain atoms from six of the eight β-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error. The mode of FMN-binding in flavocytochrome b 2 is very similar to that in two other FMN-containing enzymes, trimethylamine dehydrogenase and glycolate oxidase. All three enzymes are β 8 α 8 proteins. The detailed polypeptide conformation of the flavin-binding domain is very similar to glycolate oxidase, with which it is 37% identical in chemical sequence. However, the conformation differs considerably from that of trimethylamine dehydrogenase (sequence unknown).

X-ray refinement study on the binding of cytidylic acid (2′-CMP) to ribonuclease A

The X-ray structure of the inhibitor complex of bovine ribonuclease A with cytidylic acid (2′-CMP) has been determined at 2.3 Å (1 Å = 0.1 nm) resolution and refined by restrained least-squares refinement to R = 0.132 for 5650 reflections. Incorporation of the inhibitor molecule has occurred with little disturbance of the protein main-chain atoms, although significant displacement of some side-chain atoms has occurred, particularly in the region of the active site. The binding of 2′-CMP to ribonuclease A is different from that of the related cytidine- N(3)-oxide 2′-phosphate, which has an extra oxygen on N(3) of the cytidine base. The PO 4 2− group is held by hydrogen bond interactions to the side-groups of His12, Glu11 and His119. Thr45 is involved in stabilizing the enzyme-ligand complex by forming hydrogen bond interactions between O(γ) and the pyrimidine base N(3) atom and between the main-chain N(45) and O(2) of the base. Phe120 is much closer to the inhibitor than in the cytidine N(3)-oxide 2′-phosphate structure.

An X-ray refinement study on the binding of ribonuclease-A to cytidine-N(3)-oxide 2′-phosphate

The X-ray structure of the inhibitor complex of bovine ribonuclease-A with cytidine-N(3)-oxide 2′-phosphate (O(3)-2′CMP) has been determined at 2.3 Å resolution and refined by restrained least squares to R = 0.195 for 5240 reflections. Little disturbance of the protein main-chain atoms has taken place, but incorporation of the inhibitor molecule is associated with significant movement of side-groups, particularly within the active site. The PO 4 2− group of the inhibitor molecule occupies the SO 4 2− site of the native protein (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38, 2210–2217), while the modified pyrimidine rng is located in the B1 region and the ribose moiety is close to site R1 (Haar, W., Maurer, W. and Rüterjans, H. (1974) Eur. J. Biochem. 44, 201–211). Hydrogen bond interactions occur between the PO 4 2− group and side-groups of His-12, Lys-41 and His-119. Lys-41 has moved from its native position to establish a direct contact with the PO 4 2− group (a water molecule being expelled in the process) and His-119 now occupies exclusively the minor native site. Thr-45 plays an important role in stabilizing the modified pyrimidine base by forming hydrogen bond interactions both between O(γ) and the base N(4) atom and the NH amide group and O(3) of the base. Phe-120 does not form any close association with the planar base of the inhibitor.

Study of the interfacial properties of water by gas chromatography

Abstract : A review of our work on the study of the interfacial properties of water by gas chromatography is presented. The theory and practice of this method is outlined, along with a critical analysis of sources of error. In this method, the retention of nonelectrolytes is measured as a function of temperature and sample size. For some solutes, adsorption at the gas-liquid interface is the only mechanism of retention, and thus one can determine thermodynamic quantities of adsorption and equations of state directly. For other solutes (e.g. aromatics) both partition and adsorption simultaneously occur, resulting in a slightly more sophisticated analysis to extract both solution and adsorption phenomena. The results indicate that water is a low energy surface to hydrocarbons. Anti-Langmuir adsorption isotherms reveal that the hydrocarbon is more attracted to itself in the liquid surface than to the water molecules. Also shown for the first time is the formation of hydrogen bond interactions between water surface molecules and Bronsted bases. (Author Modified Abstract)