Structure of vesicles formed from non-ionic dialkylglycerol poly(oxyethylene) ether surfactants: effect of electrolyte and cholesterol

Five non-ionic dialkylglycerol poly(oxyethylene) ether surfactants, designated 2C_mE_n (where m, the number of carbons in each alkyl chain = 16 or 18, and n, the number of oxyethylene units = 12, 16 or 17) have been examined for their ability to form vesicles when dispersed in water or in an aqueous solution of 154 mM NaCl, alone or in the presence of 50 mol% cholesterol. Freeze fracture electron microscopy and light scattering showed that regardless of the hydrating fluid, all the non-ionic surfactants, with the exception of 2C₁₆E₁₇ and 2C₁₈E₁₇, formed vesicles in the absence of cholesterol – 2C₁₆E₁₇ and 2C₁₈E₁₇ instead formed micellar aggregates. All surfactants, however, formed vesicles in the presence of 50 mol% cholesterol. Small angle neutron scattering studies of the surfactant vesicles enabled the bilayer thickness and repeat distance (d-spacing) to be determined. The bilayers formed by all the non-ionic surfactants in the absence of cholesterol were surprisingly thin (∼50 Å for the E₁₂ containing surfactants and ∼64 Å for 2C₁₈E₁₆) most likely due to the intrusion of oxyethylene groups into the hydrophobic core of the bilayers. In contrast, however, the non-ionic surfactants exhibited a relatively large d-spacing of around ∼130–150 Å. The addition of 50 mol% cholesterol had a dramatic effect on the thickness of the vesicle bilayer, increasing its size by 10–20 Å, most probably because of an extrusion of oxyethylene from the hydrophobic region of the bilayer and/or a reduction in the tilt on the surfactant alkyl chains. Additionally the presence of cholesterol in a vesicle tended to reduce slightly both the d-spacing and the thickness of the water layer separating the bilayers. The presence of NaCl, even at the low concentrations used in the study, did affect the properties of the bilayer such that it reduced the d-spacing and, in the case of cholesterol-containing systems, also reduced bilayer thickness.     

Purification and characterization of an N-acetyl-d-galactosamine-specific lectin from the edible mushroom Schizophyllum commune

An N-acetyl-d-galactosamine (GalNAc)-specific lectin was purified from the edible mushroom, Schizophyllum commune, using affinity chromatography on a porcine stomach mucin (PSM)-Sepharose 4B column. Under reducing and non-reducing conditions, SDS-polyacrylamide gel electrophoresis gave a major band of 31.5 kDa. The Schizophyllum commune lectin (SCL) showed high affinity toward rat erythrocytes and the sugar inhibition assay exhibited its sugar specificity highly toward lactose and N-acetyl-d-galactosamine. It was stable at 55 °C for 30 min and at pH 3–10 for 18-h test. The lectin was shown to be a glycoprotein with cytotoxic activity against human epidermoid carcinoma cells. The N-terminus of SCL was blocked but amino acid sequences of internal tryptic peptides showed moderately sequence similarities with some other fungal and plant lectins. Crystals of SCL were obtained by the sitting drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant, and gave an X-ray diffraction pattern to approximately 3.8 Å resolution.     

The Crystal Structure of the Complexes of Concanavalin A with 4′-Nitrophenyl-α-d-mannopyranoside and 4′-Nitrophenyl-α-d-glucopyranoside

Concanavalin A (Con A) is the best-known plant lectin and has importantin vitrobiological activities arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complexes of Con A with 4′-nitro-phenyl-α-d-mannopyranoside (α-PNM) and 4′-nitrophenyl-α-d-glucopyranoside (α-PNG) have been crystallized in space group P2₁2₁2 with cell dimensionsa= 135.19 Å,b= 155.38 Å,c= 71.25 Å anda= 134.66 Å,b= 155.67 Å, andc= 71.42 Å, respectively. X-ray diffraction intensities to 2.75 Å for the α-PNM and to 3.0 Å resolution for the α-PNG complex have been collected. The structures of the complexes were solved by molecular replacement and refined by simulated annealing methods to crystallographic R-factor values of 0.185/0.186 and free-R-factor values of 0.260/0.274, respectively. In both structures, the asymmetric unit contains four molecules arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site near the surface of each monomer. The nonsugar (aglycon) portion of the compounds used helps to identify the exact orientation of the saccharide in the sugar-binding pocket and is involved in major interactions between tetramers. The hydrogen bonding network in the region of the binding site has been analyzed, and only minor differences with the previously reported Con A–methyl-α-d-mannopyranoside complex structure have been observed. Structural differences that may contribute to the slight preference of the lectin for mannosides over glucosides are discussed. Calculations indicate a negative electrostatic surface potential for the saccharide binding site of Con A, which may be important for its biological activity. It is also shown in detail how a particular class of hydrophobic ligands interact with one of the three so-called characteristic hydrophobic sites of the lectins.     

The Cellulose System in the Cell Wall ofMicrasterias

The cellulose system of the cell wall ofMicrasterias denticulataandMicrasterias rotatawas analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose ofMicrasteriasis very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having ad-spacing of 0.60 nm [(110) in the Iβ cellulose unit cell defined by Sugiyamaet al.,1991,Macromolecules24, 4168–4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only inSpirogyra,another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose ofMicrasteriasmay be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes.     

Atomic force microscopy reveals aggregation of gastric mucin at low pH

Mammalian gastric mucin, at high concentration, is known to form a gel at low pH, behavior essential to the protection of the stomach from auto-digestion. Atomic force microscopy (AFM) measurements of dilute solutions of porcine gastric mucin in an aqueous environment in the pH range 6-2 provide a direct visualization of extended fiberlike molecules at pH 6 that aggregate at pH 4 and below forming well-defined clusters at pH 2. The clusters consist of 10 or less molecules. AFM images of mucin at high concentration at pH 2 reveal clusters similar to those seen in the dilute solutions at low pH. We also imaged human gastric mucus revealing a network having a "pearl necklace" structure. The "pearls" are similar in size to the clusters found in the purified porcine gastric mucin gels. AFM images of deglycosylated mucin reveal that the deglycosylated portions of the molecule re-fold into compact, globular structures suggesting that the oligosaccharide chains are important in maintaining the extended conformation of mucin. However, the oligosaccharides do not appear to be directly involved in the aggregation at low pH, as clusters of similar size are observed at pH 2 in both native and deglycosylated mucin.     

Crystal structure of the complex of concanavalin A and tripeptide

The X-ray structure analysis of a cross-linked crystal of concanavalin A soaked with the tripeptide molecule as the probe molecule showed electron density corresponding to full occupation in the binding pocket. The site lies on the surface of concanavalin A and is surrounded by three symmetry-related molecules. The crystal structure of the tripeptide complex was refined at 2.4-Å resolution to an R-factor of 17.5%, (R_free factor of 23.7%), with an RMS deviation in bond distances of 0.01 Å. The model includes all 237 residue of concanavalin A, 1 manganese ion, 1 calcium ion, 161 water molecules, 1 glutaraldehyde molecule, and 1 tripeptide molecule. This X-ray structure analysis also provides an approach to mapping the binding surface of crystalline protein with a probe molecule that is dissolved in a mixture of organic solvent with water or in neat organic solvent but is hardly dissolved in aqueous solution.     

Crystal structures of 26 kDa Clonorchis sinensis glutathione S-transferase reveal zinc binding and putative metal binding

The crystal structures of CsGST in two different space groups revealed that Asp26 and His79 coordinate a zinc ion. In one space group, His46 of an adjacent molecule participates in the coordination within 2.0 Å. In the other space group, Asp26, His79 and a water molecule coordinate a zinc ion. The CsGST–D26H structure showed that four histidine residues – His26 and His79 from one molecule and the same residues from a symmetry-related neighboring molecule – coordinate a zinc ion. The coordinated zinc ions are located between two molecules and mediate molecular contacts within the crystal.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d = 6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 °C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 °C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Synthesis, crystal structure and optical properties of a new lead bismuth borate

A new phase of the compound PbBiBO₄ has been successfully synthesized via the standard solid-state reaction, and the crystal structure has been determined from X-ray monocrystal diffraction data. It crystallizes in orthorhombic space group Cmca (No. 64) with lattice constants a = 10.782(3) Å, b = 10.502(3) Å, c = 7.477(2) Å, Z = 8. The crystal structure is characterized by a three-dimensional structure consisting of BiO₆ octahedra, PbO₄ tetrahedra and BO₃ triangles. The BiO₆ octahedra compose the layers through the O(2) atoms and O(3) atoms along the [1 0 1] direction, which connects the BO₃ trigonal groups by sharing the O(3) and two O(1) atoms and links the PbO₄ tetrahedra through the O(2) atoms. The UV–Vis–NIR Diffuse-Reflectance spectrum shows that the ultraviolet cutoff edge for the PbBiBO₄ crystal is about 310 nm. Functional groups in the sample were identified by Infrared spectrum.     

Role of hydroxyl group on the mesomorphism of alkyl glycosides: synthesis and thermal behavior of alkyl 6-deoxy-β-d-glucopyranosides

A homologous series of alkyl 6-deoxy-β-d-glucopyranoside amphiphiles was prepared, in an effort to identify the role of hydroxyl group in the mesomorphic behavior of alkyl glycosides. Synthesis was performed by a chlorination of the sugar moiety in alkyl β-d-glucopyranosides with methylsulfonyl chloride in DMF, followed by a metal mediated dehalogenation to secure alkyl 6-deoxy-β-d-glucopyranosides, wherein the alkyl chain length varied from C₉ to C₁₆. The mesomorphic behavior of these 6-deoxy alkyl glycosides was assessed using polarizing optical microscopy, differential scanning calorimetry and X-ray diffraction method. Whereas the lower homologues exhibited a monotropic SmA phase till sub-ambient temperatures, the higher homologues formed a plastic phase. A partial interdigitized bilayer structure of SmA phase is inferred from experimental d-spacing and computationally derived lengths of the molecules. The results were compared with those of normal alkyl glucopyranosides, retained with hydroxyl groups at C-2–C-6 carbons, and alkyl 2-deoxy-glucopyranosides, devoid of a hydroxyl group at C-2 and the comparison showed important differences in the mesomorphic behavior.     

Haemagglutinin of the antarctic seaweed Georgiella confluens (Reinsch) Kylin: isolation and partial characterization

The marine red alga Georgiella confluens collected from Mackellar Inlet, King George Island, South Shetland Islands, Antarctic, was used in the isolation of a protein with agglutinating activity. The Georgiella confluens haemagglutinin (GCH) was extracted with 20 mM phosphate buffer, pH 7.0, and purified through ion exchange chromatography, followed by affinity chromatography on immobilized porcine stomach mucin. Among the erythrocytes analysed (human A, B and O groups, rabbit and chicken), GCH agglutinated specifically chicken erythrocytes. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the haemagglutinin revealed a single band of 21.5 kDa, while by gel filtration on Sephadex G-100 its native molecular mass was 25.5 kDa, suggesting that GCH is a monomeric protein. Haemagglutination studies showed that the GCH activity was stable through temperature variations and did not exhibit divalent cation dependence. Furthermore, the haemagglutinin was inhibited by the complex glycoproteins of porcine stomach mucin and fetuin, whereas the mono-, di-, and trisaccharides tested showed no effect.     

The boxing glove shape of subunit <Emphasis Type="Italic">d</Emphasis> of the yeast V-ATPase in solution and the importance of disulfide formation for folding of this protein

The low resolution structure of subunit d (Vma6p) of the Saccharomyces cerevisiae V-ATPase was determined from solution X-ray scattering data. The protein is a boxing glove-shaped molecule consisting of two distinct domains, with a width of about 6.5 nm and 3.5 nm, respectively. To understand the importance of the N- and C-termini inside the protein, four truncated forms of subunit d (d _11–345, d _38–345, d _1–328 and d _1–298) and mutant subunit d, with a substitution of Cys329 against Ser, were expressed, and only d _11–345, containing all six cysteine residues was soluble. The structural properties of d depends strongly on the presence of a disulfide bond. Changes in response to disulfide formation have been studied by fluorescence- and CD spectroscopy, and biochemical approaches. Cysteins, involved in disulfide bridges, were analyzed by MALDI-TOF mass spectrometry. Finally, the solution structure of subunit d will be discussed in terms of the topological arrangement of the V₁V_O ATPase.     

Crystal structure and microstructure of cholesteryl oleyl carbonate

The crystal structure as well as the microstructure, i.e., size and strain, of crystallites of cholesteryl oleyl carbonate was determined from X-ray powder diffraction data. The X-ray line broadening was analyzed through the refinement of TCH-pseudo-Voigt function parameters (isotropic effects) and the refinement of multipolar functions, i.e., symmetrized cubic harmonics (anisotropic effects). The crystal structure turns out to be primitive monoclinic, space group Pc, type I monolayer having two molecules per unit cell with parameters: a = 18.921 ± 0.006 Å, b = 12.952 ± 0.003 Å, c = 9.276 ± 0.002 Å and β = 91.32 ± 0.03°. The average size of a well ground specimen of crystallites was 60 nm. The average micro-strain, e.g., 45 × 10⁻⁴ has been tentatively attributed to fatty chain conformational disorder. The unit cell parameters, including the lamellar thickness, of COC crystal is very closely similar to those of another, structurally similar cholesterol ester, e.g., cholesteryl oleate (CO) crystal, space group P2₁, type II monolayer. Type I monolayer structure has been established for COC on the basis of the intensity calculations of the XRD profiles of both CO and COC. The dipolar and structural disorder in a 4:1 molar, binary mixture of CO and COC can be accommodated in an induced smectic phase with a lamellar thickness, which is nearly equal to that of pure CO or pure COC.     

New intercalated layer silicate nanocomposites based on synthesized starch-g-PCL prepared via solution intercalation and in situ polymerization methods: As a comparative study

Starch-g-polycaprolacton (Starch-g-PCL) nanocomposites have been prepared with graft polymerization through in situ ring-opening polymerization of ε-caprolacton in the presence of starch and Sn(Oct)₂ (Tin(II) 2-ethyl hexanoate) as an initiator/catalyst. A surface-modified montmorillonite by dimethyl (hydrogenated tallow alkyl) ammonium cation, was used. In fact, the related nanocomposites prepared via two methods in solution and in situ with introducing different amount of loading clay. The effect of swelling time on d-spacing of silicate layers was investigated and the obtained nanocomposites were analyzed using X-ray diffraction technique. The morphology of the synthesized nanocomposites examined using Scanning Electron Microscopy (SEM) and also the thermal degradation behavior of the prepared nanocomposites accomplished with using TGA.     

Molecular Mechanics Simulations in Structure Analysis of Intercalate VOPO4·2CH3CH2OH

Molecular mechanics simulations using Cerius² combined with X-ray diffraction and supported with vibrational spectroscopy have been used to investigate the layered structure of vanadyl phosphate VOPO₄ intercalated with ethanol. This intercalated structure exhibits certain degree of disorder, which affects the diffraction diagram and obstructs the conventional structure analysis based on diffraction methods only. Present structure analysis is focused to the crystal packing in the interlayer space and layer stacking in the intercalate. The bilayer arrangement of ethanol molecules in the interlayer has been found, giving the basal spacing d = 13.21 Å, experimental d-value obtained from X-ray diffraction is 13.17 Å. One half from the total number of CH₃CH₂OH molecules is anchored with their oxygens to VOPO₄ layers to complete vanadium octahedra and their orientation is not very strictly defined. The second half of ethanoles is linked with hydrogen bridges to the anchored etahanoles and sometimes also to the layer oxygens. Positions and orientations of these unachored ethanoles with respect to VOPO₄ layers exhibit certain degree of disorder, resulting in the disorder in layer stacking. Molecular mechanics simulations revealed the character of this displacement disorder in layer stacking and enabled to determine the components of the displacement vector.     

On the complex formation of iron(III) bromide in the space-demanding solvent N,N′-dimethylpropyleneurea and the structure of the trisbromoiron(III) complex in solution and crystalline state

The complex formation between iron(III) and bromide has been studied calorimetrically in N,N′-dimethylpropyleneurea (DMPU), and the structure of the DMPU solvated tribromoiron(III) complex has been studied in solution by extended X-ray absorption fine structure (EXAFS) and large angle X-ray scattering (LAXS), and in solid state by EXAFS and single crystal X-ray diffraction. The calorimetric study showed that iron(III) forms three medium strong bromide complexes in DMPU, and the thermodynamic pattern strongly indicates that all complexes are formed in entropy driven substitution reactions. In DMPU solution, the tribromoiron(III) complex has a regular trigonal planar configuration with a mean Fe-Br bond distance of 2.36 Å, and without any solvent molecules strongly bound to iron(III). In the solid state, however, the structure is a slightly distorted trigonal bipyramid, with one short and two slightly longer Fe-Br bonds, 2.37 and 2.44 Å, respectively, in a somewhat distorted trigonal plane, and two DMPU solvent molecules (mean Fe-O bond distance 1.98 Å) in the apical positions. The DMPU solution of iron(III) bromide and the [FeBr₃(dmpu)₂] crystals are both blackish red.     

Effect of citric acid and processing on the performance of thermoplastic starch/montmorillonite nanocomposites

A facile two steps extrusion processing conditions are used to prepare thermoplastic starch (TPS)/glycerol modified-montmorillonite (GMMT) nanocomposites. X-ray diffraction (XRD) and transmission electron microscopy (TEM) demonstrate glycerol can enlarge the d-spacing and destruct the multilayer structure of montmorillonite (MMT) effectively using high speed emulsifying machine (HSEM) in the first modification step. So the enlarged d-spacing and destructed platelets of MMT are favorable to form intercalated or exfoliated TPS/GMMT nanocomposites in the second melt extrusion processing. However, scanning electron microscopy (SEM) and XRD show the possible competition between TPS matrix and plasticizer for the intercalation between MMT layers can deteriorate the plasticization of TPS. In addition, citric acid (CA) can increase the plasticization of TPS and dispersion of MMT in nanocomposites effectively detected by fourier transform infrared (FT-IR) spectroscopy and SEM. At the same time, this facile processing conditions and CA can improve the mechanical properties and water vapor permeability (WVP) of TPS/GMMT nanocomposites obviously.     

The [FeFe]-hydrogenase maturation protein HydF contains a H-cluster like [4Fe4S]-2Fe site

Formation of the catalytic six-iron complex (H-cluster) of [FeFe]-hydrogenase (HydA) requires its interaction with a specific maturation protein, HydF. Comparison by X-ray absorption spectroscopy at the Fe K-edge of HydF from Clostridium acetobutylicum and HydA1 from Chlamydomonas reinhardtii revealed that the overall structure of the iron site in both proteins is highly similar, comprising a [4Fe4S] cluster (Fe–Fe distances of ∼2.7 Å) and a di-iron unit (Fe–Fe distance of ∼2.5 Å). Thus, a precursor of the whole H-cluster is assembled on HydF. Formation of the core structures of both the 4Fe and 2Fe units may require only the housekeeping [FeS] cluster assembly machinery of the cell. Presumably, only the 2Fe cluster is transferred from HydF to HydA1, thereby forming the active site.     

Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 Å resolution

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 Å resolution by X-ray crystallography. Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built. This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32. The protein is structured as a compact 10-stranded--barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule. The NH₂-terminus portion of the molecule contains two short -helices. The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods.