Effect of calcium ions on the organization of iota-carrageenan helices: an X-ray investigation

X-ray fiber diffraction analysis confirms that calcium iota-carrageenan forms a threefold, right-handed, half-staggered, parallel, double helix of pitch 26.42 A stabilized by interchain hydrogen bonds. According to the detailed structural results, three helices are packed in a trigonal unit cell (a=23.61 and c=13.21 A). Strong interactions between the sulfate groups of neighboring helices, mediated by calcium ions and water molecules, are responsible for stabilizing the three-dimensional structure.     

X-ray structure of a sodium salt of digeneaside isolated from red alga Ceramium botryocarpum

X-ray diffraction analysis has been recently used to determine the crystal structure of the floridoside (2-O-α-d-galactopyranosylglycerol) isolated from red alga Palmaria palmata and Dilsea carnosa, respectively [Simon-Colin, C.; Michaud, F.; Léger, J.-M.; Deslandes, E. Carbohydr. Res.2003, 338, 2413-2416; Vonthron-Senechau, C.; Sopkova-de Oliveira Santos. J.; Mussio, I.; Rusig, A. M. Carbohydr. Res. 2008, 343, 2697-2698]. In this present study, a similar analysis was performed on another compound belonging to the glycopyranosyl-glycerols family present in red algae, digeneaside. The crystal structure of a hydrated sodium salt of digeneaside (sodium 2-O-α-d-mannopyranosyl-d-glycerate monohydrate) was determined by single-crystal X-ray diffraction analysis at 110 ± 3 K. The space group is C2 with Z = 4, a = 17.9315(12), b = 6.2693(4), c = 10.7805(7) Å, beta = 90.746(7)°.     

Molecular structure of the rhamsan-like exocellular polysaccharide RMDP17 from Sphingomonas paucimobilis.

X-Ray diffraction analysis of the sodium salt of the polysaccharide RMDP17, a 2-deoxy rhamsan analog, reveals that it adopts a gellan-like, half-staggered, threefold, left handed, double helix of pitch 57.4 A. The side chain of the branched polymer is hydrogen bonded to the main chain. Sodium ions, linked to the carboxylate groups, promote the association of helices via water molecules. Two helices of opposite polarity occupy a trigonal unit cell of dimensions a=17.6 and c=28.7 A. The packing arrangement displays a series of hydrogen bonds involving main chain and side chain atoms, as well as some water bridges, between the helices.     

X-ray diffraction analysis of dehydrated myelin

Improved X-ray diffraction data from dry nerve myelin are presented. In addition to the spacings of approx. 150 Å, 60 Å, 44 Å and 34.6 Å, which have been previously reported, we identify a 14 Å series. The data suggests that the hydrocarbon chains in the single bilayer ($\text{≈ 60}\text{A}\text{?}$) is ordered, whereas in the double bilayer ($\text{≈ 150}\text{A}\text{?}$) and in the fluid phase ($\text{≈ 44}\text{A}\text{?}$) it is disordered. It is shown that cholesterol ($\text{≈34.6}\text{A}\text{?}$) exists as a bilayer, and the 14 Å series is probably another cholesterol phase.     

X-ray crystal structure of iridoid glucoside aucubin and its aglycone

X-ray diffraction analyses of iridoid glycoside aucubin (1) and its aglycone aucubigenin (2) are reported. It was found that crystals of 1 are orthorhombic, with P2₁2₁2₁ space group, both cyclopentane ring and pyran ring adopt envelope conformations, and the Glc moiety is in the ⁴C₁ conformation. Crystals of 2 are monoclinic, with space group P2₁, the cyclopentane and pyran rings also adopt the envelope conformation. The absolute configurations of 1 and 2 were also determined. Intensive O–HO hydrogen bonds in both crystal lattices were observed.     

X-ray structure of the dipotassium salt of D-mannose 1-phosphate 3.25 hydrate

The first crystal structure of mannose 1-phosphate is described. The dipotassium hydrate salt crystallizes in the P2(1)2(1)2 space group. There are two independent dianions (I and II) in the asymmetric unit, which are alpha anomers adopting the 4C(1) chair conformation. The main difference between the two mannose 1-phosphate dianions is the orientation of the phosphate group with relation to the pyranosyl ring. In I, one of the phosphate oxygen atoms is antiperiplanar positions with respect to carbon atom C-1, whereas the two others are situated synclinally. The corresponding orientations of the terminal phosphate oxygen atoms in II are synperiplanar and anticlinal. The potassium cations are six- and seven-coordinate, mainly with O atoms of hydroxyl groups and water molecules. There are potassium channels extending along the c-axis. In the packing arrangement, water molecules and mannose phosphate groups also define two different types of layers parallel to a-axis. Within water channels there are extensive hydrogen-bonding networks.     

Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotB_C2) and Vibrio alginolyticus (PomB_C5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotB_C2, the dimeric conformation of the PomB_C5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.     

X-ray diffraction study of the cornea

Low-angle X-ray diffraction patterns have been recorded from the cornea. A fibre diagram was obtained: the reflections from the axial period of collagen were on the equator while reflections from the collagen fibril lattice structure were on the meridian. Only the reflections from tha array of collagen fibrils have been studied. These reflections contain a primary first-order reflection and up to four subsidiary maxima. The first-order reflection from the array provides an estimate of the interfibril separation distance. Evidence is presented that the subsidiary maxima are consistent with the intensity transform of a uniform cylinder with a constant radius. Values for the fibril diameters and the interfibril distances are obtained for corneas from rabbit, cow and frog and from corneas of two marine fishes: toadfish and skate. Although the volume fraction of the collagen fibrils cannot be directly evaluated, an upper limit can be given. Thus, an upper limit of 0.28 was obtained for rabbit cornea.     

X-ray observations on a collagen fibril lattice structure in peripheral nerve

It has been known for more than two decades that peripheral nerve shows X-ray reflections other than those originating from the myelin sheath. These extra reflections are at small angles of diffraction and arise from a variation of electron density in the radial direction. X-ray diffraction studies since 1973 have identified these reflections as coming from a lattice structure of filaments, the filament axes are parallel to the nerve fibre axis. The present X-ray observations were obtained using a high-resolution X-ray camera and we conclude that these reflections arise from a collagen fibril lattice structure within peripheral nerve. Estimates of fibril radius and the separation distance between fibrils in intact rat, rabbit and frog sciatic nerves have been obtained using X-ray analysis.     

Heterogeneity in iota-carrageenan molecular structure: insights for polymorph II-->III transition in the presence of calcium ions

Iota-carrageenan is used in pharmaceutical and food applications due to its ability to complex with other hydrocolloids and proteins. Six distinct cation dependent allomorphs, consistent with its versatile functionality, have so far been observed in the solid state. In this contribution, X-ray structural details of calcium iota-carrageenan (form III) are reported. The polysaccharide retains the half-staggered, parallel, 3-fold, right-handed double helix stabilized by interchain hydrogen bonds from O-2H and O-6H in the Galp units. Results show that there are four helices, rather than one in I or three in II, organized in a larger pseudo-trigonal unit cell of dimensions a=27.44, c=13.01 A, and gamma=120 degrees . The four helices have similar core structures, but their sulfate group orientations are quite different. Fifteen calcium ions and 64 water molecules hold the helices together and promote helix-helix interactions. The results portray how the helices would shuffle around in an orchestrated manner to yield calcium iota-carrageenan III from II.     

Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy

Crystalline structures of cellulose (named as Cell 1), NaOH-treated cellulose (Cell 2), and subsequent CO2-treated cellulose (Cell 2-C) were analyzed by wide-angle X-ray diffraction and FTIR spectroscopy. Transformation from cellulose I to cellulose II was observed by X-ray diffraction for Cell 2 treated with 15-20 wt% NaOH. Subsequent treatment with CO2 also transformed the Cell 2-C treated with 5-10 wt% NaOH. Many of the FTIR bands including 2901, 1431, 1282, 1236, 1202, 1165, 1032, and 897 cm(-1) were shifted to higher wave number (by 2-13 cm(-1)). However, the bands at 3352, 1373, and 983 cm(-1) were shifted to lower wave number (by 3-95 cm(-1)). In contrast to the bands at 1337, 1114, and 1058 cm(-1), the absorbances measured at 1263, 993, 897, and 668 cm(-1) were increased. The FTIR spectra of hydrogen-bonded OH stretching vibrations at around 3352 cm(-1) were resolved into three bands for cellulose I and four bands for cellulose II, assuming that all the vibration modes follow Gaussian distribution. The bands of 1 (3518 cm(-1)), 2 (3349 cm(-1)), and 3 (3195 cm(-1)) were related to the sum of valence vibration of an H-bonded OH group and an intramolecular hydrogen bond of 2-OH ...O-6, intramolecular hydrogen bond of 3-OH...O-5 and the intermolecular hydrogen bond of 6-O...HO-3', respectively. Compared with the bands of cellulose I, a new band of 4 (3115 cm(-1)) related to intermolecular hydrogen bond of 2-OH...O-2' and/or intermolecular hydrogen bond of 6-OH...O-2' in cellulose II appeared. The crystallinity index (CI) was obtained by X-ray diffraction [CI(XD)] and FTIR spectroscopy [CI(IR)]. Including absorbance ratios such as A1431,1419/A897,894 and A1263/A1202,1200, the CI(IR) was evaluated by the absorbance ratios using all the characteristic absorbances of cellulose. The CI(XD) was calculated by the method of Jayme and Knolle. In addition, X-ray diffraction curves, with and without amorphous halo correction, were resolved into portions of cellulose I and cellulose II lattice. From the ratio of the peak area, that is, peak area of cellulose I (or cellulose II)/total peak area, CI(XD) were divided into CI(XD-CI) for cellulose I and CI(XD-CII) for cellulose II. The correlation between CI(XD-CI) (or CI(XD-CII)) and CI(IR) was evaluated, and the bands at 2901 (2802), 1373 (1376), 897 (894), 1263, 668 cm(-1) were good for the internal standard (or denominator) of CI(IR), which increased the correlation coefficient. Both fraction of the absorbances showing peak shift were assigned as the alternate components of CI(IR). The crystallite size was decreased to constant value for Cell 2 treated at >or= 15 wt% NaOH. The crystallite size of Cell 2-C (cellulose II) was smaller than that of Cell 2 (cellulose I) treated at 5-10 wt% NaOH. But the crystallite size of Cell 2-C (cellulose II) was larger than that of Cell 2 (cellulose II) treated at 15-20 wt% NaOH.     

Crystallization and preliminary X-ray crystallographic analysis of ImmE7 protein of colicin E7

The ImmE7 protein, which can bind specifically to the DNase colicin E7 and neutralize its bactericidal activity, has been purified and crystallized in two different crystal forms by vapor diffusion method. The orthorhombic crystals belong to space group I222 or I2₁2₁2₁ and have unit cell dimensions a = 75.1 Å, b = 50.5 Å, and c = 45.4 Å. The second form is monoclinic space group P2₁ with ceil dimensions a = 29.3 Å, b = 102.7 Å, c = 53.0 Å and β = 91.5°. The orthorhombic crystals diffract to 1.8 Å resolution, and are suitable for high-resolution X-ray analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

X-ray diffraction analysis of internal wool lipids

Polarised optical microscopy (POM) and X-ray diffraction techniques were applied to intercellular lipids extracted from wool to study their structural arrangement in order to determine their role in the diffusion properties of wool fibre. Intercellular wool lipids (IWL) arranged as concentrated liposomes were shown to be a good intercellular lipid model, allowing their study by X-ray diffraction techniques. The results confirm that intercellular lipids of wool fibre are organised in a lamellar structure of 5.0–8.0 nm width, termed β-layer, which had been assumed to be lipids arranged as a bilayer. Structurally, internal wool lipids are distributed at least in two domains at low temperatures: an ordered phase made up of ceramides and free fatty acids (FFA) alone, arranged in crystal orthorhombic states separately, and a liquid crystal state when mixed together. At 40 °C there is a reversible phase transition produced by the melt of the crystal orthorhombic states, whereas the liquid crystal state remains until 65 °C.     

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.     

X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium,potassium and calcium cations

Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na⁺ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds.     

X-ray structure of Glu 53 human lysozyme.

The three-dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 A resolution by X-ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild-type HL. The root mean square difference for the superposition of equivalent C alpha atoms is 0.141 A. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild-type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 A between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild-type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.     

The structure of the Mn4Ca2+ cluster of photosystem II and its protein environment as revealed by X-ray crystallography

The location, structure and protein environment of the Mn4Ca2+ cluster, which catalyses the light-driven, water-splitting reaction of photosystem II, has been revealed by X-ray crystallography. However, owing to the low resolutions of the crystal structures reported to date, and the possibility of radiation damage at the catalytic centre, the precise position of each metal ion remains unknown. To some extent, these problems have been overcome by applying spectroscopic techniques like extended X-ray absorption fine structure. Taking into account the most recent results obtained with these two X-ray-based techniques, we have attempted to refine models of the structure of the Mn4Ca2+ cluster and its protein environment.     

Crystallinity of plant epicuticular waxes: electron and X-ray diffraction studies

The crystal structure of the epicuticular waxes of 35 plant species has been examined by electron diffraction and X-ray powder diffraction. The waxes include the most common morphological wax types such as platelets, tubules, films and rodlets. Most of them were prepared with a special mechanical isolation method, which preserves the original crystal structure. Solvent-extracted recrystallized plant waxes were compared with mechanically isolated samples. The waxes were found to occur in three different crystal structures. Most of the waxes exhibited an orthorhombic structure which is the most common for aliphatic compounds. Tubules containing mainly secondary alcohols showed diffraction reflections of a triclinic phase; broad reflection peaks indicated a significant disorder. Ketones, in particular beta-diketone tubules, displayed the reflections of a hexagonal structure. Mixtures of different phases could be identified. For most of the waxes, the 'long spacing' diffraction reflections indicated a layer structure with the characteristics of the major component. Others showed no 'long spacing' reflections indicating a strong disorder of the molecular layers.     

Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.