Structural characterization of tissue-specific galactan from flax fibers by 1H NMR and MALDI TOF mass spectrometry

A high-molecular-mass polysaccharide galactan (M 2000 kDa) was isolated from flax at the stage of cell wall thickening of the bast fiber development. The polymer structure was studied by ¹H NMR spectroscopy and MALDI TOF mass spectrometry. It is built up of Gal (59%), Rha (15%), GalA (23%), and Ara (3%) residues. The galactan backbone consists of successively alternating monomer disaccharide units (→ 4GalA1 → 2Rha1 →)_n and is similar in its structure to the backbone of rhamnogalacturonan-1 (RG-I). Rhamnose residues bear in position 4 β-(1 → 4)-galactose side chains of various lengths with a polymerization degree of up to 28 or higher. A part of the side chains have branchings.     

Molecular and crystal structure of the regenerated form of (1→3)-α-d-glucan

The crystal structure of a regenerated form of (1→3)-α-d-glucan, obtained by solid state deacetylation of the triacetate derivative, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. The structure crystallizes in an orthorhombic unit cell with parameters $\text{a = 16.46}\text{A}\text{?}\text{, b = 9.55}\text{A}\text{?}$ and c (fibre repeat)=8.44 Å, and space group P2₁2₁2₁. The chain conformation is nearly completely extended and is very close to a 2/1 helix, even though the dimer residue is the crystallographic repeat unit. An intramolecular O(2)  O(4)′ hydrogen bond stabilizes the conformation and extensive intermolecular hydrogen-bonding abilizes the packing. The resulting structure is sheet-like, with an alternating polarity of chain directions within the sheet. In its sheet-like character, extensive hydrogen-bonding, and insolubility in water, this polymorph of (1→3)-α-d-glucan resembles regenerated cellulose. The reliability of the structure analysis is indicated by the X-ray residual R=0.206.     

Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers: X-ray diffraction study

The structural changes accompanying the recently described sub-transition of hydrated dipalmitoylphosphatidylcholine (Chen, S.C., Sturtevant, J.M. and Gaffney, B.J. (1980) Proc. Natl. Acad. Sci. USA 77, 5060–5063) have been defined using X-ray diffraction methods. Following prolonged storage at ?4°C the usual L_β′ gel form of hydrated dipalmitoylphosphatidylcholine (DPPC) is converted into a more ordered stable ‘crystal’ form. The bilayer periodicity is 59.1 Å and the most striking feature is the presence of a number of X-ray reflections in the wide angle region. The most prominent of these are a sharp reflection at $\text{1}\text{4.4}\text{A}\text{?}{}^{\mathrm{?1}}$ and a broader reflection at $\text{1}\text{3.9}\text{A}\text{?}{}^{\mathrm{?1}}$. This diffraction pattern is indicative of more ordered molecular and hydrocarbon chain packing modes in this low temperature ‘crystal’ bilayer form. At the sub-transition (T_rmsub = 15–20°C) an increase in the bilayer periodicity occurs ($\text{d=63.6}\text{A}\text{?}$) and a strong reflection at approx. $\text{1}\text{4.2}\text{A}\text{?}{}^{\mathrm{?1}}$ with a shoulder at approx. $\text{1}\text{4.1}\text{A}\text{?}{}^{\mathrm{?1}}$ is observed. This diffraction pattern is identical to that of the bilayer gel (L_β′) form of hydrated DPPC. Thus, the sub-transition corresponds to a bilayer ‘crystal’ → bilayer L_β′ gel structural rearrangement accompanied by a decrease in the lateral hydrocarbon chain interactions. Differential scanning calorimetry and X-ray diffraction show that on further heating the usual structural changes L_β′ → P_β′ and P_β′ → L_α occur at the pre- and main transitions, at approx. 35°C and 41°C, respectively.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

Purification and structural properties of the precursors of the globin messenger RNAs

X-ray diffraction data have been obtained from sodium and calcium salts of a proteoglycan rich in chondroitin 4-sulfate isolated from the Swarm rat chondrosarcoma. When sodium is the only countercation associated with the proteoglycan, the oriented polysaccharide chains adopt a 3-fold helical conformation in the solid state and pack in a trigonal unit cell with dimensions a = b = 1.45 nm and c = 2.88 nm. Addition of small amounts of calcium or full conversion of the polyanion from a sodium to a calcium salt form results in a conformational transition to a somewhat more extended 2-fold structure.For the calcium salt X-ray intensity data were used to refine the polysaccharide conformation and packing arrangement in the unit cell. Two antiparallel chains were found to crystallize in an orthorhombic unit cell with space group P22₁2₁ and dimensions a = 0.745 nm, b = 1.781 nm and c = 1.964 nm. The individual helix axes intersect the base plane of the unit cell at (x_f = 0, y_f = 0) and ($\text{x}{}_{\mathrm{f}}\text{= 0, y}{}_{\mathrm{f}}\text{=}\text{1}\text{2}$), and the polyanions are crystallographically equivalent, being related by the symmetry of the space group.The conformation of chondroitin 4-sulfate is stabilized intramolecularly by O.3 … O.5 hydrogen bonds across the β(1 → 4) linkage as well as by OSO^?₃ … Ca²⁺ … ^?OOC co-ordination across the β(1 → 3) linkage. Within the lattice adjacent parallel chains interact through COO^? … Ca²⁺ … ^?OOC bridges, and each calcium co-ordination shell is completed with an additional five water molecules to form a distorted, square antiprism. These water molecules are hydrogen-bonded to neighboring polyanions, and all intermolecular interactions involve water bridges or calcium ion co-ordination.On the basis of the refined packing model and the known structural features of the proteoglycan, models are considered for proteoglycan organization in connective tissue. Consideration of the conformational directing influence and relative abundance of calcium in the intercellular matrix suggest that the secondary structure of chondroitin 4-sulfate in vivo is likely to be similar to the conformation described in this study.     

Crystal structure analysis of sea lamprey hemoglobin at 2 Å resolution

The crystal structure of the predominant hemoglobin component of blood from the sea lamprey, Petromyzon marinus, has been determined by X-ray diffraction analysis. Crystals for this analysis were grown from cyanide methemoglobin V as crystal type D₂. These crystals are in space group P2₁2₁2₁ and have unit cell dimensions of $\text{a = 44.57}\text{A}\text{?}\text{, b = 96.62}\text{A}\text{?}\text{and}\text{c = 31.34}\text{A}\text{?}$. Isomorphous heavyatom derivatives were prepared by soaking crystals in solutions of Hg(CN)₂, K₂Hg(CNS)₄ and KAu(CN)₂. Diffracted intensities to as far as 2 Å spacings were measured on a diffractometer. Phases were found by means of the isomorphous replacements and anomalous scattering, with supplementary information provided by the tangent formula. An atomic model was fitted to the final electron density map in a Richards optical comparator. The lamprey hemoglobin molecule is generally similar in structure to other globins, but differs in many details. Each molecule is in contact with ten neighboring molecules in the crystal lattice. The nature of the binding of the heavy atoms to lamprey hemoglobin has been interpreted.     

Mass spectrometry and 13C nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins

The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H₂O and D₂O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-β-d-glucopyranosylamine, and 1-N-acetyl-2-acetamido-β-d-glucopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at $\text{m}\text{e}\text{= 269}$, no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the α-O-glycosidic linkage, fully assigned carbon NMR spectra of α-methyl-N-acetylgalactosamine (GalNAc), α-methyl-3-O-methyl GalNAc,and -GlcNAc as well as the disaccharide Glc-β-1 → 3 GalNAc are reported. Because certain anomalies in the chemical shifts and ¹J_CH observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides.     

Polysaccharides of basidiomycetes. Alkali-soluble polysaccharides from the mycelium of white rot fungus <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> (Curt.: Fr.) P. Karst

Two polysaccharides were isolated from submergedly cultured mycelium of the basidiomycete Ganoderma lucidum by extraction with alkali followed by fractionation with Fehling reagent. The polysaccharides were shown to be a linear (1→3)-α-D-glucan and a highly branched xylomannan containing a backbone built up of (1→3)-linked α-D-mannopyranose residues, the majority of which are substituted at O-4 by single β-D-xylopyranose residues or by disaccharide fragments β-D-Manp-(1→3)-β-D-Xylp-(1→. Polysaccharide structures were elucidated by NMR spectroscopy in combination with methylation analysis and periodate oxidation. An interesting feature of the xylomannan is the simultaneous presence of α-D-mannopyranose and β-D-mannopyranose residues, the first forming the backbone, and the second being the non-reducing terminal units of disaccharide side chains.     

Preparation,crystal structure and thermal behaviour of bischromatodihydroxobis-(pyridine)tricopper(II) [Cu3(CrO4)2(OH)2(C5H5N)2]

The title compound was prepared by slow crystallization from a hot aqueous solution of copper(II)- dichromate and pyridine. The structure determination was performed at room temperature on a single crystal in the triclinic space group P$\text{1}$, a = 5.378(1), b = 5.619(1), c = 13.569(2) Å, α = 93.32(1), β = 100.25(1), γ=98.45(1)°. Using 2026 reflections with F_o² > (F_o²) obtained on a CAD-4 single crystal diffractometer the structure was solved by conventional Patterson and Fourier methods and full matrix least-squares refinement to R = 0.047. The structure consists of complex chains built up from two different (4 + 2) distorted copper(II) octahedra sharing common edges. These chains are linked via OCrO bonds thus forming a two-dimensional infinite network. The pyridine rings extending into the space between these layers are disordered due to rotation around the CuN bond. In the course of the refinement two favoured positions with occupation probabilities 50:50 percent were found. During thermal decomposition the compound loses pyridine and water followed by a release of oxygen to yield poly- crystalline CuCr₂O₄ and CuO. An intermediate phase with empirical formula Cu₃O(CrO₄)₂ was detected by X-ray powder diffraction and its unit cell parameters were determined.     

Crystallographic study of the anti-tumor protein ricin

The anti-tumor protein ricin (also called RCA_II) has been crystallized in a form suitable for X-ray diffraction analysis. The crystal symmetry is orthorhombic spacegroup P2₁2₁2₁ with $\text{a = 72.9, b = 79.1, c = 114.7}\text{A}\text{?}$ and a single ricin molecule of molecular weight 65,000 per asymmetric unit. The crystals diffract well and are stable in the X-ray beam. At least one useful heavymetal derivative has been found as part of a high-resolution study.     

Particle and crystal symmetry of erysimum latent virus

Erysimum latent virus, a tymovirus, contains 180 protein subunits arranged in a T = 3 icosahedral surface lattice. A cubic crystal form (with space group P2₁3 and $\text{a = 414}\text{A}\text{?}$) and a monoclinic form (space group B2, $\text{a = 442}\text{A}\text{?}\text{, b = 422}\text{A}\text{?}\text{, c = 387}\text{A}\text{?}\text{, γ = 95 °}$) have been observed. The asymmetric units of the two crystal forms contain one-third and one whole virus particle, respectively. Two possible packing arrangements of the virus particles in the monoclinic unit cell have been deduced from the low-angle diffraction patterns. X-ray diffraction data from the monoclinic crystals extend to at least 3·7 Å resolution.     

Interactions and space requirement of the phosphate head group of membrane lipids: The single crystal structures of a triclinic and a monoclinic form of hexadecyl-2-deoxyglycerophosphoric acid monohydrate

The crystal structures of a triclinic form (HPA1) and a monoclinic form (HPA2) of hexadecyl-2-deoxyglycerophosphoric acid monohydrate were determined by single crystal analysis. The unit cell dimensions for HPA1 are $\text{a = 4.75, b = 5.72, c = 44.36}\text{A}\text{?}$ and α = 91.0, β = 101.5, γ = 100.5° (P$\text{1}$) and for HPA2, $\text{a = 4.75, b = 5.72, c = 88.72}\text{A}\text{?}$ and γ = 100.8° (P2₁). In both structures the molecules are fully extended and pack tail-to-tail in bilayers with tilting (47°) hydrocarbon chains. In HPA2, however, the chain tilt alternatingly changes direction in adjacent bilayers, giving rise to a doubled unit cell which spans two bilayers. The dihydrogen phosphate groups interact by hydrogen bonds and are arranged in rows. Laterally between these phosphate rows the water molecules are accommodated producing a compact two-dimensional network of hydrogen bonds. The packing cross-section in the layer plane of the dihydrogen phosphate monohydrate group is 26.7 Ų in both structures. The hydrocarbon chains pack according to the triclinic (T|) chain packing mode. In HPA2, however, the chain packing is somewhat less compact with accounts for a 2% increase in the molecular volume. In both structures the ether oxygen is accommodated into the hydrocarbon matrix without distortion of the chain packing.     

Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

Simulation of the conformational flexibility of the mycodextran under external forces

Mycodextran—also known as nigeran—is an unbranched polysaccharide made of α-d -glucopyranose units alternatively connected by (1 → 3) and (1 → 4) glycosidic linkages produced intracellularly by Aspergillus niger and Penicillium crustosum. In this work we examine possible enforced conformational transitions in the glucopyranose rings in the nigeran oligosaccharide chains. In order to simulate such structural changes we used the Enforced Geometry Optimization (EGO) method.     

High resolution nuclear magnetic resonance studies of nigeran

Polymer motion in solution can be studied by ¹³CNMR relaxation methods, which provide information about the correlation time for C-H vectors. ¹³C-Relaxation and Nuclear Overhauser Enhancement (NOE) data may frequently be combined to determine the dipole-dipole relaxation contribution. An alternative method is proposed based on a comparison of the proton spin-lattice relaxation rates of the centre proton resonances of an unlabelled molecule with the relaxation rates of the ¹³C satellites (from ¹³C labelled molecules).Selectively labelled nigeran which is an alternating $\text{1 → 3 and 1 → 4 α-}\text{d}\text{-glucan}$ has been investigated. The discussion in terms of the occurrence of different motions for each of the two units of the polymer requires an unambiguous assignment of the two anomeric carbons. For this reason a detailed assignment of the ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra of nigeran in dimethylsulphoxide-d₆ is described, based on T₁ and NOE measurements in addition to selective homonuclear and heteronuclear spin decoupling experiments. These values are correlated with a conformation estimated by HSEA hard-spheres calculation. The measurements of the relaxation parameters for labelled and unlabelled compounds which provide an alternative determination of the ¹³C-¹H dipole-dipole relaxation contribution in a macromolecule agree well with ¹³C-{¹H} NOE experiments.     

Structural components of alginic acid. I. The crystalline structure of poly-beta-D-mannuronic acid. Results of x-ray diffraction and polarized infrared studies

A Structure determination of the naturally occuring marine algal polysaccharide poly-β-D -mannuronic acid is described. The structure consists of 1e → 4e linked D -mannuronic acid chains with the monosaccharide units in the C1 chair conformation. The X-ray fiber diffraction photograph obtained from bundles of fibers prepared from Fucus vesiculosus has been indexed to an orthorhombic unit cell in which a =7.6 Å, b (fiber axis) = 10.4 Å, c = 8.6 Å, the unit cell containing two disaccharide chain segments with space group P2₁2₁2₁. A sheet-like structure involving one intra-chain, one intra-sheet, and one inter-sheet hydrogen bond per monosaccharide is proposed. Features of the chain-packing arrangement are compared with mannan.     

Corrections to Nomenclature of Phosphorus-Containing Compounds of Biochemical Importance

Crystals of cholesteryl oleate (C₄₅H₇₈O₂) are monoclinic, space group P2₁, with $\text{a = 12.65(3), b = 9.13(3), c = 18.79(5)}\text{A}\text{?}\text{, β = 93.3(3)°}$ and have 2 molecules in the unit cell. The crystal structure has been determined by Patterson and Fourier methods at a resolution $\text{d}{}_{\mathrm{<mtext>min</mtext>}}\text{= 1.1}\text{A}\text{?}$, using 799 X-ray intensities (CuKα) measured by a diffractometer. Structure refinement by block-diagonal least squares gave R = 0.12. The oleate chains are almost straight except for a kink at the cis-double bond. The chains pack side by side but without a regular sub-cell structure, in a manner which might be similar to the arrangement within biological membranes. As in cholesteryl octanoate, the cholesteryl ring systems pack together with extensive overlap of anti-parallel nearest neighbours. Projecting methyl groups interlock.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk β-N-acetylglucosaminide β1 → 4-galactosytransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [¹⁴C]galactosylated product chain was obtained as a reduced oligosaccharide by β-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is $\text{Gal}\text{β1 → 3([}{}^{14}\text{C}\text{]}\text{Gal}\text{β1 → 4}\text{GlcNac}\text{β1 → 6)}\text{GalNAcol}$. Thus it appears that Galβ1 → 3(GlcNAcβ1 → 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of α-N-acetylgalactosaminylprotein β1 → 3-galactosyltransferase activity using ovine submaxillary asialomucin as an receptor can be counteracted by the addition of N-acetylglucosamine.     

Iota-carrageenan: molecular structure and packing of polysaccharide double helices in oriented fibres of divalent cation salts

The Ca²⁺ and Sr²⁺ salts of i-carrageenan have isomorphous crystal structures with trigonal unit cells of dimensions a = b = 1.373 nm, c = 1.328 nm, γ = 120 °. Two kinds of fibre diffraction pattern were found for the Mg²⁺ salt: one resembling the Ca²⁺ and Sr²⁺ patterns and one with additional layer lines interleaved midway between those in the usual kind of pattern. Specimens of this second type convert to the first type on storage at 92% relative humidity. These Mg²⁺i-carrageenate diffraction patterns provide direct evidence for the double-helical nature of the carrageenan molecule.A molecular model has been derived that consists of two, identical, righthanded, 3-fold helical polysaccharide chains of pitch 2.656 nm. One chain is translated axially 1.32 nm relative to the other.A packing arrangement with up-pointing and down-pointing double helices distributed randomly among the molecular sites explains the presence of both Bragg reflections and layer line streaks. The space group of our statistical crystal structure is P3₂12. The divalent cations were found by Fourier difference syntheses to be at ($\text{2}\text{3}$, $\text{1}\text{3}$, $\text{1}\text{6}$) and symmetry-related positions. The co-ordination of each cation to sulphate groups on two different helices leads to a continuous set of cation-sulphate-cation-… interactions that accounts for the high crystallinity of these salts.The structure of the Ca²⁺ salt has been refined by constrained linked-atom least-squares methods. The structural isomorphism of the Sr²⁺ salt was confirmed by an independent refinement.     

IS50-mediated inverse transposition. Discrimination between the two ends of an IS element

The crystal and molecular structure of l-pyroglutamyl-β-(2-thienyl)-l-alanyl-l-prolinamide, < Glu-Thi-Pro-NH₂(Thi²-TRH), C₁₇H₂₂N₄O₄S, has been determined from X-ray diffraction data. Thi²-TRH is a highly active analogue of thyroliberin, a thyrotropin-releasing hormone (TRH), in which the imidazole ring of the central histidine moiety in the natural hormone has been replaced by a 2-thienyl group. Thi²-TRH crystallizes from water in the monoclinic space group P2₁, $\text{a = 9.340(1)}\text{A}\text{?}\text{, b = 21.961(3)}\text{A}\text{?}\text{, c = 9.449(1)}\text{A}\text{?}$ and β = 109.58(1) °, with two molecules per asymmetric unit. These independent molecules, A and B, have the same general backbone conformation with the φ₂, ψ₂ and ψ₃ torsional angles close to ?90 °, +120 ° and +150 °, respectively, but they show different magnitudes of rotational disorder in the thiophene ring as well as a certain disorder in the pyrrolidine ring. A and B are cross-linked by four interchain hydrogen bonds, forming a two-stranded antiparallel β-pleated sheet structure. The molecules in these dimer fragments are further hydrogen-bonded to successive translated molecules along the a and c axes, forming a pronounced two-dimensional predominantly hydrophobic layer structure. These layers, in which the atoms are almost equally arranged on both sides, are separated by ordinary van der Waals' distances. A close correlation between the molecular conformation in the solid state and the preferential conformation in solution is found. It is concluded that the crystalline structure of Thi²-TRH possesses structural features which may be of relevance in the hormone-receptor interaction process.