Synthesis and crystal structure of an octamer RNA r(guguuuac)/r(guaggcac) with G.G/U.U tandem wobble base pairs: comparison with other tandem G.U pairs

We have determined the crystal structure of the RNA octamer duplex r(guguuuac)/r(guaggcac) with a tandem wobble pair, G·G/U·U (motif III), to compare it with U·G/G·U (motif I) and G·U/U·G (motif II) and to better understand their relative stabilities. The crystal belongs to the rhombohedral space group R3. The hexagonal unit cell dimensions are a = b = 41.92 Å, c = 56.41 Å, and γ = 120°, with one duplex in the asymmetric unit. The structure was solved by the molecular replacement method at 1.9 Å resolution and refined to a final R factor of 19.9% and R_free of 23.3% for 2862 reflections in the resolution range 10.0–1.9 Å with F ≥ 2σ(F). The final model contains 335 atoms for the RNA duplex and 30 water molecules. The A-RNA stacks in the familiar head-to-tail fashion forming a pseudo-continuous helix. The uridine bases of the tandem U·G pairs have slipped towards the minor groove relative to the guanine bases and the uridine O2 atoms form bifurcated hydrogen bonds with the N1 and N2 of guanines. The N2 of guanine and O2 of uridine do not bridge the ‘locked’ water molecule in the minor groove, as in motifs I and II, but are bridged by water molecules in the major groove. A comparison of base stacking stabilities of motif III with motifs I and II confirms the result of thermodynamic studies, motif I > motif III > motif II.     

Protein and drug interactions in the minor groove of DNA

Interactions between proteins, drugs, water and B-DNA minor groove have been analyzed in crystal structures of 60 protein–DNA and 14 drug–DNA complexes. It was found that only purine N3, pyrimidine O2, guanine N2 and deoxyribose O4′ are involved in the interactions, and that contacts to N3 and O2 are most frequent and more polar than contacts to O4′. Many protein contacts are mediated by water, possibly to increase the DNA effective surface. Fewer water-mediated contacts are observed in drug complexes. The distributions of ligands around N3 are significantly more compact than around O2, and distributions of water molecules are the most compact. Distributions around O4′ are more diffuse than for the base atoms but most distributions still have just one binding site. Ligands bind to N3 and O2 atoms in analogous positions, and simultaneous binding to N3 and N2 in guanines is extremely rare. Contacts with two consecutive nucleotides are much more frequent than base–sugar contacts within one nucleotide. The probable reason for this is the large energy of deformation of hydrogen bonds for the one nucleotide motif. Contacts of Arg, the most frequent amino acid ligand, are stereochemically indistinguishable from the binding of the remaining amino acids except asparagine (Asn) and phenylalanine (Phe). Asn and Phe bind in distinct ways, mostly to a deformed DNA, as in the complexes of TATA-box binding proteins. DNA deformation concentrates on dinucleotide regions with a distinct deformation of the δ and backbone torsion angles for the Asn and δ, , ζ and χ for the Phe-contacted regions.     

Crystal structure of the complementary quadruplex formed by d(GCATGCT) at atomic resolution

Here we report the crystal structure of the DNA heptanucleotide sequence d(GCATGCT) determined to a resolution of 1.1 Å. The sequence folds into a complementary loop structure generating several unusual base pairings and is stabilised through cobalt hexammine and highly defined water sites. The single stranded loop is bound together through the G(N2)–C(O2) intra-strand H-bonds for the available G/C residues, which form further Watson–Crick pairings to a complementary sequence, through 2-fold symmetry, generating a pair of non-planar quadruplexes at the heart of the structure. Further, four adenine residues stack in pairs at one end, H-bonding through their N7–N6 positions, and are additionally stabilised through two highly conserved water positions at the structural terminus. This conformation is achieved through the rotation of the central thymine base at the pinnacle of the loop structure, where it stacks with an adjacent thymine residue within the lattice. The crystal packing yields two halved biological units, each related across a 2-fold symmetry axis spanning a cobalt hexammine residue between them, which stabilises the quadruplex structure through H-bonds to the phosphate oxygens and localised hydration.     

Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)₂ duplex. Moreover, the D positions in 27 D₂O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.     

Intermolecular interactions and water structure in a condensed phase B-DNA crystal

By controlled dehydration, the unit cells of dodecamer DNA–drug crystals have been shrunk from 68 000 (normal state) to 60 000 (partially dehydrated intermediate state) to 51 000 ų (fully dehydrated state), beyond which no further solvent loss occurs. The total solvent content in the normal crystals is ~40% by volume, reducing to ~20% in the fully dehydrated phase. The 25% reduction in cell volume induced a dramatic enhancement in the resolution of the X-ray diffraction data (from 2.6 to beyond 1.5 Å). We have determined the structures of the normal, partially dehydrated and fully dehydrated crystals. Details of the ligand binding have been presented in the preceding article. The present paper describes the unique features of the structure of the fully dehydrated phase. This structure was refined with 9015 unique observed reflections to R = 14.9%, making it one of the most reliable models of B-form DNA available. The crystals exist as infinite polymeric networks, in which neighbouring dodecamer duplexes are crosslinked through phosphate oxygens via direct bonding to magnesium cations. The DNA is packed so tightly that there is essentially only a single layer of solvent between adjacent molecules. The details of the crystal packing, magnesium bridging, DNA hydration and DNA conformation are described and compared with other experimental evidence related to DNA condensation.     

Crystal Structure of Reaction Intermediates in Pyruvate Class II Aldolase: SUBSTRATE CLEAVAGE,ENOLATE STABILIZATION,AND SUBSTRATE SPECIFICITY*

Crystal structures of divalent metal-dependent pyruvate aldolase, HpaI, in complex with substrate and cleavage products were determined to 1.8–2.0 Å resolution. The enzyme·substrate complex with 4-hydroxy-2-ketoheptane-1,7-dioate indicates that water molecule W2 bound to the divalent metal ion initiates C3–C4 bond cleavage. The binding mode of the aldehyde donor delineated a solvent-filled capacious binding locus lined with predominantly hydrophobic residues. The absence of direct interactions with the aldehyde aliphatic carbons accounts for the broad specificity and lack of stereospecific control by the enzyme. Enzymatic complex structures formed with keto acceptors, pyruvate, and 2-ketobutyrate revealed bidentate interaction with the divalent metal ion by C1-carboxyl and C2-carbonyl oxygens and water molecule W4 that is within close contact of the C3 carbon. Arg⁷⁰ assumes a multivalent role through its guanidinium moiety interacting with all active site enzymatic species: C2 oxygen in substrate, pyruvate, and ketobutyrate; substrate C4 hydroxyl; aldehyde C1 oxygen; and W4. The multiple interactions made by Arg⁷⁰ stabilize the negatively charged C4 oxygen following proton abstraction, the aldehyde alignment in aldol condensation, and the pyruvate enolate upon aldol cleavage as well as support proton exchange at C3. This role is corroborated by loss of aldol cleavage ability and pyruvate C3 proton exchange activity and by a 730-fold increase in the dissociation constant toward the pyruvate enolate analog oxalate in the R70A mutant. Based on the crystal structures, a mechanism is proposed involving the two enzyme-bound water molecules, W2 and W4, in acid/base catalysis that facilitates reversible aldol cleavage. The same reaction mechanism promotes decarboxylation of oxaloacetate.     

G-quadruplexes with (4n?- 1) guanines in the G-tetrad core: formation of a G-triad·water complex and implication for small-molecule binding

G-quadruplexes are non-canonical structures of nucleic acids, in which guanine bases form planar G-tetrads (G·G·G·G) that stack on each other in the core of the structure. G-quadruplexes generally contain multiple times of four (4n) guanines in the core. Here, we study the structure of G-quadruplexes with only (4n - 1) guanines in the core. The solution structure of a DNA sequence containing 11 guanines showed the formation of a parallel G-quadruplex involving two G-tetrads and one G-triad with a vacant site. Molecular dynamics simulation established the formation of a stable G-triad·water complex, where water molecules mimic the position of the missing guanine in the vacant site. The concept of forming G-quadruplexes with missing guanines in the core broadens the current definition of G-quadruplex-forming sequences. The potential ability of such structures to bind different metabolites, including guanine, guanosine and GTP, in the vacant site, could have biological implications in regulatory functions. Formation of this unique binding pocket in the G-triad could be used as a specific target in drug design.     

Crystal structure of 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide bound to d(CGTACG)2: implications for structure–activity relationships of acridinecarboxamide topoisomerase poisons

The structure of the complex formed between d(CGTACG)₂ and 9-amino-N-[2-(4-morpholinyl)ethyl]-4-acridinecarboxamide, an inactive derivative of the antitumour agents N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and 9-amino-DACA, has been solved to a resolution of 1.8 Å using X-ray crystallography. The complex crystallises in the space group P6₄ and the final structure has an overall R factor of 21.9%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and its protonated morpholino nitrogen partially occupying positions close to the N7 and O6 atoms of guanine G2. The morpholino group is disordered, the major conformer adopting a twisted boat conformation that makes van der Waals contact with the O4 oxygen of thymine T3. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of guanine G2. Sugar rings are found in alternating C3′-exo/C2′-endo conformations except for cytosine C1 which is C3′-endo. Intercalation perturbs helix winding throughout the hexanucleotide compared with B-DNA, steps 1 and 2 being unwound by 10 and 8°, respectively, while the central TpA step is overwound by 11°. An additional drug molecule lies at the end of each DNA helix linking it to the next duplex to form a continuously stacked structure. The protonated morpholino nitrogen of this ‘end-stacked’ drug hydrogen bonds to the N7 atom of guanine G6, and its conformationally disordered morpholino ring forms a C–H···O hydrogen bond with the guanine O6 oxygen. In both drug molecules the 4-carboxamide group is internally hydrogen bonded to the protonated N10 atom of the acridine ring. We discuss our findings with respect to the potential role played by the interaction of the drug side chain and the topoisomerase II protein in the poisoning of topoisomerase activity by the acridinecarboxamides.     

Lone pair ··· π interactions between water oxygens and aromatic residues: Quantum chemical studies based on high‐resolution protein structures and model compounds

The π electron cloud of aromatic centers is known to be involved in several noncovalent interactions such as C—H···π, O—H···π, and π···π interactions in biomolecules. Lone-pair (lp) ··· π interactions have gained attention recently and their role in biomolecular structures is being recognized. In this article, we have carried out systematic analysis of high-resolution protein structures and identified more than 400 examples in which water oxygen atoms are in close contact (distance < 3.5 Å) with the aromatic centers of aromatic residues. Three different methods were used to build hydrogen atoms and we used a consensus approach to find out potential candidates for lp···π interactions between water oxygen and aromatic residues. Quantum mechanical calculations at MP2/6-311++G(d,p) level on model systems based on protein structures indicate that majority of the identified examples have energetically favorable interactions. The influence of water hydrogen atoms was investigated by sampling water orientations as a function of two parameters: distance from the aromatic center and the angle between the aromatic plane and the plane formed by the three water atoms. Intermolecular potential surfaces were constructed using six model compounds representing the four aromatic amino acids and 510 different water orientations for each model compound. Ab initio molecular orbital calculations at MP2/6-311++G(d,p) level show that the interaction energy is favorable even when hydrogen atoms are farthest from the aromatic plane while water oxygen is pointing toward the aromatic center. The strength of such interaction depends upon the distance of water hydrogen atoms from the aromatic substituents. Our calculations clearly show that the lp···π interactions due to the close approach of water oxygen and aromatic center are influenced by the positions of water hydrogen atoms and the aromatic substituents.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.     

Cyclic diguanylic acid behaves as a host molecule for planar intercalators

Cyclic ribodiguanylic acid, c-(GpGp), is the endogenous effector regulator of cellulose synthase. Its three-dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X-ray diffraction analysis at 1 A resolution. In both crystal forms, two independent c-(GpGp) molecules associate with each other to form a self-intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32 degrees), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c-(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage-like model consisting of a tetrameric c-(GpGp) aggregate in which a large cavity ('host') is generated to afford a binding site for certain planar intercalators ('guests').     

Crystal and molecular structure of the ammonium salt of the dinucleoside monophosphate d(CpG)

The crystal and molecular structure of the ammonium salt of deoxycytidylyl-(3'-5')-deoxyguanosine has been determined from 0.85 A resolution single crystal X-ray diffraction data. The crystals obtained by acetone diffusion technique at -20 degrees C, are orthorhombic, P212121, a = 12.880(2), b = 17444(2) and c = 27.642(2) A. The structure was solved by high resolution Patterson and Fourier methods and refined to R = 0.136. There are two d(CpG) molecules in the asymmetric unit forming a mini left handed Z-DNA helix. This is in contrast to the earlier reported forms of d(CpG) where the molecules form self base paired duplexes. There are two ammonium ions in the asymmetric unit. The major groove NH+4 ion interacts with N7 of guanines through water bridges besides making H-bonded interactions directly with the phosphate oxygen atoms. A second NH+4 ion is found in the minor groove interacting directly with the phosphate oxygen atoms. Symmetry related molecules pack in such a way that the cytosine base stacks on cytosine and guanine base on guanine. Our structure demonstrates that alternating d(CpG) sequences have the ability to adopt the left handed Z-DNA structure even at the dimer level i.e., in a sequence which is only two base pairs long.     

Hydration of transfer RNA molecules: a crystallographic study

Four crystal structures of transfer RNA molecules were refined at 3 A resolution with the inclusion of the solvent molecules found in the difference maps: yeast tRNA-phe in the orthorhombic form, yeast tRNA-phe in the monoclinic form and yeast tRNA-asp in the A and B forms. Over 100 solvent molecules were located in each tRNA crystal. Several hydration schemes are found repeatedly in the 4 crystals. The tertiary interactions in the corner of the L-shaped molecule attract numerous solvent molecules which bridge the ribose hydroxyl O(2') atoms, base exocyclic atoms and phosphate anionic oxygen atoms. Conservation of bases leads to conservative localized hydration patterns. Several solvent molecules are found stabilizing unusual base pairs like the G-U pairs and those involving the pseudouridine base. Water bridges between the O(2') and the exocyclic atom O2 of pyrimidines or the N3 atom of purines are common. Water bridges occur frequently between successive anionic oxygen atoms of each strand as well as between N7 or other exocyclic atoms of successive bases in the major groove. Magnesium ions or spermine molecules are found to bind in the major groove of tRNA helices without specific interactions.     

Analysis of solvent structure in proteins using neutron D2O-H2O solvent maps: pattern of primary and secondary hydration of trypsin.

A method of determining the water structure in protein crystals is described using neutron solvent difference maps. These maps are obtained by comparing the changes in diffracted intensities between two data sets, one in which H2O is the major solvent constituent, and a second in which D2O is the solvent medium. To a good first approximation, the protein atom contributions to the scattering intensities in both data sets are equal and cancel, but since H2O and D2O have very different neutron-scattering properties, their differences are accentuated to reveal an accurate representation of the solvent structure. The method also employs a series of density modification steps that impose known physical constraints on the density distribution function in the unit cell by making real space modifications directly to the density maps. Important attributes of the method are that (1) it is less subjective in the assignment of water positions than X-ray analysis; (2) there is threefold improvement in the signal-to-noise ratio for the solvent density; and (3) the iterative density modification produces a low-biased representation of the solvent density. Tests showed that water molecules with as low as 10% occupancy could be confidently assigned. About 300 water sites were assigned for trypsin from the refined solvent density; 140 of these sites were defined in the maps as discrete peaks, while the remaining were found within less-ordered channels of density. There is a very good correspondence between the sites in the primary hydration layer and waters found in the X-ray structure. Most water sites are clustered into H-bonding networks, many of which are found along intermolecular contact zones. The bound water is equally distributed between contacting apolar and polar atoms at the protein interface. A common occurrence at hydrophobic surfaces is that apolar atoms are circumvented by one or more waters that are part of a larger water network. When the effects on surface accessibility by neighboring molecules in the crystal lattice are taken into consideration, only about 29% of the surface does not interface ordered water. About 25% of the ordered water is found in the second hydration sphere. In many instances these waters bridge larger clusters of primary layer waters. It is apparent that, in certain regions of the crystal, the organization of ordered water reflects the characteristics of the crystal environment more than those of trypsin's surface alone.     

Structurally conserved water molecules in ribonuclease T1

In the high resolution (1.7-1.9 A) crystal structures of ribonuclease T1 (RNase T1) in complex with guanosine, guanosine 2'-phosphate, guanylyl 2',5'-guanosine, and vanadate, there are 30 water sites in nearly identical (+/- 1 A) positions that are considered conserved. One water is tightly bound to Asp76(O delta), Thr93(O gamma), Cys6(O), and Asn9(N); another bridges two loops by hydrogen-bonding to Tyr68(O eta) and to Ser35(N), Asn36(N); a loop structure is stabilized by two waters coordinated to Gly31(O) and His27(N delta), and by water bound to cis-Pro39(O). Most notable is a hydrogen-bonded chain of 10 water molecules. Waters 1-5 of this chain are inaccessible to solvent, are anchored at Trp59(N), and stitch together the loop formed by segments 60-68; waters 5-8 coordinate to Ca2+, and waters 9 and 10 hydrogen-bond to N-terminal side chains of the alpha-helix. The water chain and two conserved water molecules are bound to amino acids adjacent to the active site residues His40, Glu58, Arg77, and His92; they are probably involved in maintaining their spatial orientation required for catalysis. Water sites must be considered in genetic engineering; the mutation Trp59Tyr, which probably influences the 10-water chain, doubles the catalytic activity of RNase T1.     

Variability in the backbone conformation of cyclic pentapeptides. Crystal structure of cyclic(Gly-L-Pro-D-Phe-Gly-L-Ala)

All the peptide bonds in cyclic(Gly-L-Pro-D-Phe-Gly-L-Ala) are in the trans conformation; however, the peptide bond C'5-N1 is twisted by 19 degrees from planarity (omega 5 = -161 degrees). A Type II beta-turn encompasses the L-Pro-D-Phe residues. Carbonyl oxygens O2, O4 and O5 are directed to the same side of the average plane through the backbone ring and they form hydrogen bonds with N3, N5 and N1, respectively, in adjacent molecules in a stacked column where the adjacent molecules are related by one translational unit. The conformation of the backbone is different from that established in other molecules with the DLDDL chirality sequence. The P21 cell contains two molecules of C21H26N5O5 with a = 4.836(2) A, b = 18.346(8) A, c = 12.464(5) A and beta = 100.05(4) degrees. The R factor for 1382 data with [F0[ greater than 1 sigma is 7.0%.     

1 A crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium

The 1 A resolution X-ray crystal structures of Mg(2+) and Ca(2+) salts of the B-DNA decamers CCAACGTTGG and CCAGCGCTGG reveal sequence-specific binding of Mg(2+) and Ca(2+) to the major and minor grooves of DNA, as well as non-specific binding to backbone phosphate oxygen atoms. Minor groove binding involves H-bond interactions between cross-strand DNA base atoms of adjacent base-pairs and the cations' water ligands. In the major groove the cations' water ligands can interact through H-bonds with O and N atoms from either one base or adjacent bases, and in addition the softer Ca(2+) can form polar covalent bonds bridging adjacent N7 and O6 atoms at GG bases. For reasons outlined earlier, localized monovalent cations are neither expected nor found.Ultra-high atomic resolution gives an unprecedented view of hydration in both grooves of DNA, permits an analysis of individual anisotropic displacement parameters, and reveals up to 22 divalent cations per DNA duplex. Each DNA helix is quite anisotropic, and alternate conformations, with motion in the direction of opening and closing the minor groove, are observed for the sugar-phosphate backbone. Taking into consideration the variability of experimental parameters and crystal packing environments among these four helices, and 24 other Mg(2+) and Ca(2+) bound B-DNA structures, we conclude that sequence-specific and strand-specific binding of Mg(2+) and Ca(2+) to the major groove causes DNA bending by base-roll compression towards the major groove, while sequence-specific binding of Mg(2+) and Ca(2+) in the minor groove has a negligible effect on helix curvature. The minor groove opens and closes to accommodate Mg(2+) and Ca(2+) without the necessity for significant bending of the overall helix.The program Shelxdna was written to facilitate refinement and analysis of X-ray crystal structures by Shelxl-97 and to plot and analyze one or more Curves and Freehelix output files.     

Biological Macromolecular Dynamics

Abstract

Experimentally observed sequence-selective binding of metal ion to DNA oligonucleotides have been compared with variations of electrostatic potential (EP) along the helix. Calculations of EP have been performed for three atomic models of the oligonucleotide duplex [d(CGCGAATTCGCG)2] using several variants of EP calculations, including a solution of non-linear Poisson-Boltzmann equation (NPBE). N7 atom of guanine adjacent to adenine base was identified as a region with the most negative electrostatic potential in the major groove. The EP value for the Me ion binding site surpasses the value for N7 of other guanines by 10–26% depending on particular duplex conformation. Qualitatively, the sequence dependent variations of EP near guanine N7 atoms are in agreement with the sequence-selective behavior of Mn(II) and Zn(II) ions as revealed by NMR experiments. But the difference in EP between the two most negative regions near guanine N7 atoms does not exceed 1.25 kT/e. Simple model suggests that metal ions are capable to form ion-hydrate complexes with G-Pu steps of DNA duplex. These complexes are formed via one Me…G and five Me…water coordination bonds with water molecules hydrogen bonded to two adjacent purine bases in the same chain. We suppose that such a stereospecific structural possibility is the main factor which control the sequence-selectivity in the metal ion binding. A combination of both mechanisms allows to explain sequence specific Mn(II) and Zn(II) binding to a set of oligonucleotides.     

Oxygen exchange between nitrate molecules during nitrite oxidation by Nitrobacter

During oxidation of nitrite, cells of Nitrobacter winogradskyi are shown to catalyze the active exchange of oxygen atoms between exogenous nitrate molecules (production of 15N16/18O3- during incubation of 14N16/18O3-, 15N16O3-, and 15N16O2- in H216O). Little, if any, exchange of oxygens between nitrate and water also occurs (production of 15N16/18O3- during incubation of 15N16O3- and 14N16O2- in H218O). 15N species of nitrate were assayed by 18O-isotope shift in 15N NMR. Taking into account the O-exchange reactions which occur during nitrite oxidation, H2O is seen to be the source of O in nitrate produced by oxidation of nitrite by N. winogradskyi. The data do not establish whether the nitrate-nitrate O exchange is catalyzed by nitrite oxidase (H2O + HNO2----HNO3 + 2H+ + 2e-) or nitrate reductase (HNO3 + 2H+ + 2e-----HNO2 + H2O) or both enzymes in consort. The nitrate-nitrate exchange reaction suggests the existence of an oxygen derivative of a H2O-utilizing oxidoreductase.     

Direct determination of the hydrogen bonding arrangement in anhydrous β-chitin by neutron fiber diffraction

The hydrogen bonding arrangement in anhydrous β-chitin, a homopolymer of N-acetylglucosamine, was directly determined by neutron fiber diffraction. Data were collected from a sample prepared from the bathophilous tubeworm Lamellibrachia satsuma in which all labile hydrogen atoms had been replaced by deuterium. Initial positions of deuterium atoms on hydroxyl and acetamide groups were directly located in Fourier maps synthesized using phases calculated from the X-ray structure and amplitudes measured from the neutron data. The hydrogen bond arrangement in the refined structure is in general agreement with predictions based on the X-ray structure: O3 donates a hydrogen bond to the O5 ring oxygen atom of a neighboring residue in the same chain; N2 and O6 donate hydrogen bonds to the same carbonyl oxygen O7 of an adjacent chain. The intramolecular O3···O5 hydrogen bond has the most energetically favorable geometry with a hydrogen to acceptor distance of 1.77 ? and a hydrogen bond angle of 171°.