The structure of the asparagine-linked sugar chains of bovine brain ribonuclease

The asparagine-linked sugar chains of bovine brain ribonuclease were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. After N-acetylation, they were converted into radioactively-labeled oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharide mixture was fractionated by ion-exchange chromatography, and the acidic oligosaccharides were converted into neutral oligosaccharides by sialidase digestion. The neutral oligosaccharides were then fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion in combination with methylation analysis revealed that bovine brain ribonuclease showed extensive heterogeneity. It contains bi- and tri-antennary, complex-type oligosaccharides having alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D- GlcpNAc-(1----4)-[alpha-L-Fucp-(1----6)]-D-GlcNAc as their common core. Four different outside oligosaccharide chains, i.e., beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----6)-beta-D- Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAc-(1----, and alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, were found. The preferential distribution of the alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc group on the alpha-D-Manp-(1----6) arm is a characteristic feature of the sugar chains of this enzyme.     

The structure of polymer chains in confinement. A Monte Carlo study

A coarse-grained model of polymer star chains confined in two parallel impenetrable surfaces, which were attractive for polymer beads was studied. The flexible homopolymer chains were built of united atoms whose positions in space were restricted to vertices of a simple cubic lattice. The chains were modeled in good solvent conditions and, thus, there were no long-range specific interactions between polymer beads—only the excluded volume was present. The influence of the polymer density and the distances between the confining surfaces on the properties of star-branched polymers was studied. It is shown that the chains adsorbed on one surface could change their position so that they swap between both surfaces with frequency depending on the size of the slit and on the density of the system only. The increase of the polymer density diminished the frequency of jumps and caused that chains became only partially adsorbed. The analysis of structural elements of chains showed that the increase of the density of the system leads to increase of the number of bridges connecting the two adsorbing surfaces, thus, the frequency of jumps between them decreases.     

The crystal structure of a nonalkenic,cyclic trimer of levoglucosenone

A single-crystal, X-ray diffraction study was performed on a nonalkenic, cyclic trimer (C₁₈H₁₈O₉, 4) of levoglucosenone, in order to confirm its chemical structure. Crystals of 4 are orthorhombic, with unit-cell parameters of a = 792.20, b = 1874.35, c = 2383.02 pm, space group P2₁2₁2₁, and z = 8. The structure was solved by direct methods, and refined by least-squares to R = 0.032, based on 2990 unique reflections. Each asymmetrical unit contains two symmetry-independent molecules of 4 and one of acetone. The previously assigned chemical structure and stereochemistry of 4 were found to be correct.     

Constructing side chains on near-native main chains for ab initio protein structure prediction

Is there value in constructing side chains while searching protein conformational space during an ab initio simulation? If so, what is the most computationally efficient method for constructing these side chains? To answer these questions, four published approaches were used to construct side chain conformations on a range of near-native main chains generated by ab initio protein structure prediction methods. The accuracy of these approaches was compared with a naive approach that selects the most frequently observed rotamer for a given amino acid to construct side chains. An all-atom conditional probability discriminatory function is useful at selecting conformations with overall low all-atom root mean square deviation (r.m.s.d.) and the discrimination improves on sets that are closer to the native conformation. In addition, the naive approach performs as well as more sophisticated methods in terms of the percentage of chi(1) angles built accurately and the all-atom r. m.s.d., between the native and near-native conformations. The results suggest that the naive method would be extremely useful for fast and efficient side chain construction on vast numbers of conformations for ab initio prediction of protein structure.     

Role of structure and glycosylation of adsorbed protein films in biolubrication

Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy), we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation of adsorbed protein films, providing an important clue to design effective therapeutics to restore biolubrication in patients with insufficient biolubrication.     

Secondary structure of proteins adsorbed onto or embedded in polyelectrolyte multilayers

The structural changes of bovine serum albumin (BSA) and hen egg white lysozyme (HEL) upon their adsorption onto the surface or their embedding into the interior of poly(allylamine hydrochloride)-(poly(styrenesulfonate) (PAH-PSS) multilayer architectures were investigated by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The presence of the polyelectrolytes seems, as previously observed for fibrinogen (J. Phys. Chem. B 2001, 105, 11906-11916), to prevent intermolecular interactions and, thus, protein aggregation at ambient temperature. The secondary structure of the proteins was somewhat altered upon adsorption onto the polyelectrolyte multilayers. The structural changes were larger when the charges of the multilayer outer layer and the protein were opposing. The adsorption of further polyelectrolyte layers onto protein-terminated architectures (i.e., embedding the proteins into a polyelectrolyte multilayer) did not cause considerable further changes in their secondary structures. The capacity of the polyelectrolyte architectures to delay the formation of intermolecular beta-sheets upon increasing temperatures was not uniform for the studied proteins. PSS in contact with HEL could largely prevent the heat-induced aggregation of HEL. In contrast, PAH had hardly any effect on the aggregation of BSA. The differences are explained on the basis of protein-polyelectrolyte interactions, affected mostly by the nature and the strength of the ionic interactions between the polyelectrolyte-protein contact surfaces.     

The crystal structure of cholesteryl dodecanoate: co-packing of steroid skeleta and hydrocarbon chains

The crystal structure of cholesteryl dodecanoate has been determined. The compound shows a co-packing of cholesterol skeleta and hydrocarbon chains. There are two molecules in the asymmetric unit both almost fully extended. The hydrocarbon chain axes are however somewhat bent in order to get a good close-packing side by side with the rigid cholesterol skeleta. The two non-symmetry related skeleta show different packing surroundings. One skeleton packs with both hydrocarbon chains and other skeleta while the other skeleton is completely surrounded by hydrocarbon chains. The latter packing is of particular interest as it is considered to indicate important packing principles in biological lipid bilayers.     

The structure of the O-glycosidic oligosaccharide chains of the major Zajdela hepatoma ascites-cell-membrane glycoprotein

Glycoprotein MII2, the major cell surface glycoprotein (molecular mass 110 kDa) of Zajdela hepatoma ascites cells, contains about 25 O-glycosidic oligosaccharide chains per molecule. They were released as oligosaccharide-alditols by alkaline borohydride treatment of MII2, and purified by gel filtration on Bio-Gel P-6 followed by high-voltage paper electrophoresis. Four oligosaccharide-alditol fractions (A-D) were obtained in relative yields of 8:6:3:3. The structure of the components of fractions A-C was determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis, to be as follows. (A) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B1) NeuAc alpha(2----3)Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B2) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (C) NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol. On the basis of sugar composition and characteristics on Bio-Gel P-6 filtration, paper electrophoresis and thin-layer chromatography, the structure of the carbohydrate component of fraction D is proposed to be as follows. (D) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol     

The crystal structure of a cyclic glycolipid reveals a carbohydrate-carbohydrate interaction interface

The crystal structure of an amphiphilic macrocycle [Velasco-Torrijos, T.; Murphy, P. V., Tetrahedron: Asymmetry2005, 16, 261-272] derived from saccharides shows extensive hydrogen-bonding networks, including participation of water, that may have relevance for the modelling of carbohydrate-carbohydrate recognition at cell-cell interfaces. The structure may provide a basis for understanding the binding of the macrocycle to hydrophobic probes.     

The structure of the keratan sulphate chains attached to fibromodulin from human articular cartilage

The small keratan sulphate proteoglycan, fibromodulin, has been isolated from pooled human articular cartilage. The main chain repeat region and the chain caps from the attached N-linked keratan sulphate chains have been fragmented by keratanase II digestion, and the oligosaccharides generated have been reduced and isolated. Their structures and abundance have been determined by high pH anion-exchange chromatography. These regions of the keratan sulphate from human articular cartilage fibromodulin have been found to have the following general structure: Significantly, both α(2-6)- and α(2-3)-linked N-acetyl-neuraminic acid have been found in the capping oligosaccharides. Fucose, which is α(1-3)-linked as a branch to N-acetylglucosamine, has also been found along the length of the repeat region and in the capping region. The chains, which have been found to be very highly sulphated, are short; the length of the repeat region and chain caps is ca. nine disaccharides. These data demonstrate that the structure of the N-linked keratan sulphate chains of human articular cartilage fibromodulin is similar, in general, to articular cartilage derived O-linked keratan sulphate chains. Further, the general structure of the keratan sulphate chains attached to human articular cartilage fibromodulin has been found to be generally similar to that of both bovine and equine articular cartilage fibromodulin. Abbreviations: KS, keratan sulphate; IEC, ion-exchange chromatography; ELISA, enzyme linked immunosorbent assay; Gal, β-D-galactose; Fuc, α-L-Fucose; GlcNAc, N-acetylglucosamine (2-acetamido-β-D-glucose); GlcNAc-ol, N-acetylglucosaminitol (2-acetamido-D-glucitol); NeuAc, N-acetyl-neuraminic acid; 6S/(6S), O-ester sulphate group on C6 present/sometimes present; NMR -nuclear magnetic resonance; HPAE, high pH anion-exchange; PED, pulsed electrochemical detection; HPLC, high performance liquid chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Crystal structure of cyclic Lys48-linked tetraubiquitin

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.