Optimizing crystal volume for neutron diffraction: D-xylose isomerase

Neutron diffraction is uniquely sensitive to hydrogen positions and protonation state. In that context structural information from neutron data is complementary to that provided through X-ray diffraction. However, there are practical obstacles to overcome in fully exploiting the potential of neutron diffraction, i.e. low flux and weak scattering. Several approaches are available to overcome these obstacles and we have investigated the simplest: increasing the diffracting volume of the crystals. Volume is a quantifiable metric that is well suited for experimental design and optimization techniques. By using response surface methods we have optimized the xylose isomerase crystal volume, enabling neutron diffraction while we determined the crystallization parameters with a minimum of experiments. Our results suggest a systematic means of enabling neutron diffraction studies for a larger number of samples that require information on hydrogen position and/or protonation state.     

Local mobility of nucleic acids as determined from crystallographic data. II. Z-form DNA

Directions and magnitudes of the local mobility of the Z-DNA hexamer duplex CpGpCpGpCpG have been determined by crystallographic refinement of anisotropic displacement parameters using the observed X-ray diffraction data. The cytidine and guanosine residues demonstrate different modes of mobility, implying that a dinucleotide is the smallest repeating unit in terms of flexibility as well as structure. Directions of librational and translational mobility of the cytidine and guanosine residues of Z-DNA are similar to those observed for the same nucleotides in B-DNA. This suggests that the local mobility of DNA is primarily determined by the individual nucleotide type and by the constraints of Watson-Crick base-pairing, rather than by helical form. Differences in the magnitudes of mobility may be responsible for some of the different physical properties of B-DNA and Z-DNA. The B to Z transition is discussed in terms of the observed flexibilities of these two helical forms.     

Molecular dynamics and free energy calculations of the B and Z forms of C8-arylguanine modified oligonucleotides

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8-tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6-31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5'-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.     

Effects of the O2' hydroxyl group on Z-DNA conformation: structure of Z-RNA and (araC)-[Z-DNA

The left-handed Z structures of two hexamers [d(CG)r(CG)d(CG) and d(CG)(araC)d(GCG)] containing ribose and arabinose residues have been solved by X-ray diffraction analysis at 1.5-A resolution. Their conformations closely resemble that of the canonical Z-DNA. The O2' hydroxyl groups of both rC and araC residues form intramolecular hydrogen bonds with N2 of the 5' guanine residue and replace the bridging water molecules in the deep groove of Z-DNA, which stabilize the guanine in the syn conformation. The araC residue can be incorporated into the Z structure readily and facilitates B to Z transition, as supported by UV absorption spectroscopic studies. In contrast, in Z-RNA the ribose of the cytidine residue is twisted in order to form the respective hydrogen bond. The potential biological roles of the modified Z-DNA containing anticancer nucleoside araC and of Z-RNA are discussed.     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.     

Polarizable Atomic Multipole X-Ray Refinement: Hydration Geometry and Application to Macromolecules

We recently developed a polarizable atomic multipole refinement method assisted by the AMOEBA force field for macromolecular crystallography. Compared to standard refinement procedures, the method uses a more rigorous treatment of x-ray scattering and electrostatics that can significantly improve the resultant information contained in an atomic model. We applied this method to high-resolution lysozyme and trypsin data sets, and validated its utility for precisely describing biomolecular electron density, as indicated by a 0.4-0.6% decrease in the R- and R_free-values, and a corresponding decrease in the relative energy of 0.4-0.8 Kcal/mol/residue. The re-refinements illustrate the ability of force-field electrostatics to orient water networks and catalytically relevant hydrogens, which can be used to make predictions regarding active site function, activity, and protein-ligand interaction energies. Re-refinement of a DNA crystal structure generates the zigzag spine pattern of hydrogen bonding in the minor groove without manual intervention. The polarizable atomic multipole electrostatics model implemented in the AMOEBA force field is applicable and informative for crystal structures solved at any resolution.     

Molecular Dynamics and Free Energy Calculations of the B and Z Forms of C8-Arylguanine Modified Oligonucleotides

Abstract

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8- tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6–31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5′-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.     

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°.     

Left-handed DNA and the synaptic pairing reaction promoted by Ustilago rec1 protein

Left-handed Z-DNA binds tightly to Ustilago rec1 protein. The binding reaction is strongly dependent on ATP, but complexes formed are rapidly dissociated by ADP. The parallel between the kinetics of Z-DNA binding and the synaptic pairing reaction leading to paranemic joint molecules suggests that formation of nascent heteroduplex structures in recombination is coupled with formation of left-handed Z-like DNA on the protein. Equilibrium and kinetic studies show that rec1 protein appears to have a strong Z-DNA binding site that binds Z-DNA 75 times tighter than the B form of the DNA. We propose that DNA with a structure approximated best by a left-handed Z-DNA conformation is a key intermediate in homologous pairing promoted by rec1 protein.     

Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

Water-DNA interactions as studied by X-ray and neutron fibre diffraction

X-ray fibre-diffraction studies indicate a high degree of stereochemical specificity in interactions between water and the DNA double helix. Evidence for this comes from data that show that the molecular conformations assumed by DNA in fibres are highly reproducible and that the hydration-driven transitions between these conformations are fully reversible. These conformational transitions are induced by varying the relative humidity of the fibre environment and hence its water content. Further evidence for stereochemical specificity comes from the observed dependence of the conformation assumed on the ionic content of the fibre and the nucleotide sequence of the DNA. For some transitions, information on stereochemical pathways has come from real-time X-ray fibre diffraction using synchrotron radiation; information on the location of water with respect to the double helix for a number of DNA conformations has come from neutron fibre diffraction. This structural information from fibre-diffraction studies of DNA is complemented by information from X-ray single-crystal studies of oligonucleotides. If the biochemical processes involving DNA have evolved to exploit the structural features observed in DNA fibres and oligonucleotide single crystals, the challenges in developing alternatives to a water environment can be expected to be very severe.     

The crystal and molecular structures of cellulose I and II

The paper describes molecular dynamics (MD) simulations on the crystal structures of the Iβ and II phases of cellulose. Structural proposals for each of these were made in the 1970s on the basis of X-ray diffraction data. However, due to the limited resolution of these data some controversies remained and details on hydrogen bonding could not be directly obtained. In contrast to structure factor amplitudes in X-ray diffraction, energies, as obtained from MD simulations, are very sensitive to the positions of the hydroxyl hydrogen atoms. Therefore the latter technique is very suitable for obtaining such structural details. MD simulations of the Iβ phase clearly shows preference for one of the two possible models in which the chains are packed in a parallel orientation. Only the parallel-down mode (in the definition of Gardner and Blackwell (1974) J Biopolym 13: 1975-2001) presents a stable structure. The hydrogen bonding consists of two intramolecular hydrogen bonds parallel to the glycosidic linkage for both chains, and two intralayer hydrogen bonds. The layers are packed hydrophobically. All hydroxymethyl group are positioned in the tg conformation. For the cellulose II form it was found that, in contrast to what seemed to emerge from the X-ray fibre diffraction data, both independent chains had the gt conformation. This idea already existed because of elastic moduli calculations and 13C-solid state NMR data. Recently, the structure of cellotetraose was determined. There appear to be a striking similarity between the structure obtained from the MD simulations and this cellotetraose structure in terms of packing of the two independent molecules, the hydrogen bonding network and the conformations of the hydroxymethyl group, which were also gt for both molecules. The structure forms a 3D hydrogen bonded network, and the contribution from electrostatics to the packing is more pronounced than in case of the Iβ structure. In contrast to what is expected, in view of the irreversible transition of the cellulose I to II form, the energies of the Iβ form is found to be lower than that of II by 1 kcal mol-1 per cellobiose. This revised version was published online in November 2006 with corrections to the Cover Date.     

Neutron Diffraction of Alpha,Beta and Gamma Cyclodextrins: Hydrogen Bonding Patterns

Abstract

Cyclodextrins (CD's) have proved useful as model systems for the study of hydrogen bonding. They are torus-shaped molecules composed of six(α), seven (β) or eight(γ) (1?4) linked glucoses. Because of their particular geometry, they are able to act as a “host” to form inclusion complexes with “guest” molecules very much like enzymes. Cyclodextrins have been shown to exert catalytic activity on suitable included-substrate molecules; they catalyze the hydrolysis of phenylacetates, of organic pyrophosphates and of penicillin derivatives. They also accelerate aromatic chlorinations and diazo coupling by means of their primary and/or secondary hydroxyl groups, so that the rates of hydrolysis are enhanced by up to a factor of 400. In order to understand the hydrogen bonding in these enzyme models, neutron diffraction data were collected to unambiguously determine the hydrogen atom positions, which could not be done from the x-ray diffraction data. α-CD has been shown to have two different structures with well-defined hydrogen bonds, one “tense” and the other “relaxed”. An “induced-fit”-like mechanism for α-CD complex formation has been proposed. Circular hydrogen bond networks have also been found for α-CD due to the energetically favored cooperative effect. β-CD with a disordered water structure possesses an unusual flip-flop hydrogen bonding system of the type O-H…H-O representing an equilibrium between two states: O-H…O?O…O. γ-CD with a disordered water structure similar to β-CD also possesses the flip-flop hydrogen bond. This study demonstrates that hydrogen bonds are operative in disordered systems and display dynamics even in the solid state.     

Detection of Z DNA binding proteins in tissue culture cells.

A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA.     

Mcg in 2030: new techniques for atomic position determination of immune complexes

The lambda-type light chain dimer from a patient (Mcg) with multiple myeloma and amyloidosis was a pioneer protein for determining the three-dimensional structures of immunoglobulins, understanding the effects of ligand binding, and exploring the use of combinatorial methods to identify novel peptides complementary to protein active sites. Despite 30 years of intense study, there are still unanswered questions about the structure of the Mcg dimer, especially with respect to positions of hydrogen atoms and solvent molecules. In the present report, we describe two techniques that will help define the roles of solvent in ligand interactions and complex formation with this immunoglobulin fragment: (1) introduction of helium as a cryogenic agent during X-ray data collection; and (2) addition of neutron diffraction analyses. These techniques should provide improved resolution, and a more accurate structure of the Mcg dimer. Resolution enhancements of 0.5 A have been achieved in preliminary experiments with cryogenic helium, as compared with the best X-ray diffraction data obtained previously. In the near future, neutron diffraction studies should produce the first hydrogen structure for the Mcg dimer and help elucidate the ligand preferences and amyloidogenic properties of this eminently useful protein.     

The Internal Organization of Mycobacterial Partition Assembly: Does the DNA Wrap a Protein Core?

Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A “DNA outside, protein inside” mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA.     

The active site protonation states of perdeuterated Toho-1 β-lactamase determined by neutron diffraction support a role for Glu166 as the general base in acylation

Room temperature neutron diffraction data of the fully perdeuterated Toho-1 R274N/R276N double mutant β-lactamase in the apo form were used to visualize deuterium atoms within the active site of the enzyme. This perdeuterated neutron structure of the Toho-1 R274N/R276N reveals the clearest picture yet of the ground-state active site protonation states and the complete hydrogen-bonding network in a β-lactamase enzyme. The ground-state active site protonation states detailed in this neutron diffraction study are consistent with previous high-resolution X-ray studies that support the role of Glu166 as the general base during the acylation reaction in the class A β-lactamase reaction pathway.     

The transition between the B and Z conformations of DNA investigated by targeted molecular dynamics simulations with explicit solvation

The transition between the B and Z conformations of double-helical deoxyribonucleic acid (DNA) belongs to the most complex and elusive conformational changes occurring in biomolecules. Since the accidental discovery of the left-handed Z-DNA form in the late 1970s, research on this DNA morphology has been engaged in resolving questions relative to its stability, occurrence, and function in biological processes. While the occurrence of Z-DNA in vivo is now widely recognized and the major factors influencing its thermodynamical stability are largely understood, the intricate conformational changes that take place during the B-to-Z transition are still unknown at the atomic level. In this article, we report simulations of this transition for the 3'-(CGCGCG)-5' hexamer duplex using targeted molecular dynamics with the GROMOS96 force field in explicit water under different ionic-strength conditions. The results suggest that for this oligomer length and sequence, the transition mechanism involves: 1), a stretched intermediate conformation, which provides a simple solution to the important sterical constraints involved in this transition; 2), the transient disruption of Watson-Crick hydrogen-bond pairing, partly compensated energetically by an increase in the number of solute-solvent hydrogen bonds; and 3), an asynchronous flipping of the bases compatible with a zipperlike progression mechanism.