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Biomedical subjects

G Widmalm

Publications and source records attributed to G Widmalm.

At least 19 recordsLinked to original sources
PubMed2001

Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1.

We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated. Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified. The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text). The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug). The enzyme associated with the JA1 phage responsible for the depolymerization of the V. cholerae O139 CPS is thus a lyase.

Bacteriophages↗
PubMed2001

Oligosaccharides display both rigidity and high flexibility in water as determined by 13C NMR relaxation and 1H,1H NOE spectroscopy: evidence of anti-phi and anti-psi torsions in the same glycosidic linkage.

The trisaccharide beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe has been investigated by molecular dynamics (MD) simulations and NMR experiments in water. 13C spin-lattice (T1) and spin-spin (T2) relaxation times, together with 1H,13C NOE data were measured at two magnetic field strengths (9.4 and 14.1 T) in a 277 K D2O solution. Relaxation data interpreted by means of the model-free formalism revealed a rigid (S2 approximately 0.9) oligosaccharide tumbling in solution. 1H,1H Cross-relaxation rates were determined at 600 MHz by 1D DPFGSE NOESY and T-ROESY experiments, which provided high quality data and subsequently proton-proton distances within the trisaccharide. The presence of anti conformers at both torsions of a glycosidic linkage is demonstrated for the first time. MD simulations were carried out to facilitate analysis of the NOE data. In total, 15 simulations-starting from five different conformational states--were performed, with production runs of up to 10 ns, resulting in 83 ns of oligosaccharide dynamics in water. anti Conformers were populated to different degrees in the simulations, especially at the phi2 torsion angle. By combining the results from the NOE experiments and the MD simulations, the anti conformers at the (1-->2)-linkage were quantified as 7% anti-phi2 and 2% anti-psi2, revealing a highly flexible trisaccharide in which large conformational changes occur. From the MD simulations, interresidue hydrogen bonding, from HO2" to O2 or O3, was significantly populated (approximately 40%) in both of the anti conformational states. The contentious issue over rigidity versus flexibility in oligosaccharides has thus been thoroughly examined, showing that the dynamics should be taken into account for a relevant description of the molecular system.

Carbohydrate Conformation↗
PubMed2001

Structural studies of the O-polysaccharide from the Escherichia coli O77 lipopolysaccharide.

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O77 has been determined. Sugar and methylation analysis together with 1H and 13C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure:-->2)-alpha-D-Manp-(1-->2)-beta-D-Manp-(1-->3)-alpha-D-GlcpNAc-(1-->6)-alpha-D-Manp-(1-->

Carbohydrate Conformation↗
PubMed2001

A conformational study of the trisaccharide beta-D-Glcp-(1-->2)[beta-D-Glcp-(1-->3)]alpha-D-Glcp-OMe by NMR NOESY and TROESY experiments, computer simulations, and X-ray crystal structure analysis.

Proton-proton cross-relaxation rates have been measured for the trisaccharide beta-D-Glcp-(l --> 2)[beta-D-Glcp-(1 --> 3)]alpha-D-Glcp-OMe in D2O as well as in D2O/[D6]DMSO 7:3 solution at 30 degrees C by means of one-dimensional NMR pulsed field gradient 1H,1H NOESY and TROESY experiments. Interatomic distances for the trisaccharide in D2O were calculated from the cross-relaxation rates for two intraresidue and three interglycosidic proton pairs, using the isolated spin-pair approximation. In the solvent mixture one intraresidue and three interglycosidic distances were derived without the use of a specific molecular model. In this case the distances were calculated from the cross-relaxation rates in combination with "model-free" motional parameters previously derived from 13C relaxation measurements. The proton-proton distances for interglycosidic pairs were compared with those averaged from Metropolis Monte Carlo and Langevin Dynamics simulations with the HSEA, PARM22, and CHEAT95 force fields. The crystal structure of the trisaccharide was solved by analysis of X-ray data. Interresidue proton pairs from the crystal structure and those observed by NMR experiments were similar. However, the corresponding proton-proton distances generated by computer simulations were longer. For the (1 --> 2) linkage the glycosidic torsion angles of the crystal structure were found in a region of conformational space populated by all three force fields, whereas for the (1 --> 3) linkage they occupied a region of low population density, as seen from the simulations.

Algorithms↗
PubMed2001

Structural studies of S-7, another exocellular polysaccharide containing 2-deoxy-arabino-hexuronic acid.

The exocellular polysaccharide S-7, a heteropolysaccharide from Azotobacter indicus var. myxogenes has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [structure: see text] The absolute configuration of the deoxyhexuronic acid was deduced from 1H NMR chemical shifts and is most likely D. Approximately two O-acetyl groups per repeating unit are present, one of which is presumably on the Rha residue. The structure bears great resemblance to another polysaccharide, recently studied, produced by Sphingomonas paucimobilis I-886.

Azotobacter↗
PubMed2001

Conformational investigation of a cyclic enterobacterial common antigen employing NMR spectroscopy and molecular dynamics simulations.

The three-dimensional structure of a cyclic enterobacterial common antigen (ECA) having four trisaccharide repeating units has been investigated by NMR spectroscopy and molecular dynamics simulations. Three different NMR parameters were determined: (a) (1)H,(1)H cross-relaxation rates from NOE experiments were used for determination of proton-proton distances; (b) trans-glycosidic (3)J(C,H) scalar coupling constants analyzed via a Karplus-type relationship provided information on torsion angles; and (c) (1)H,(13)C one-bond dipolar couplings obtained in a dilute liquid-crystalline medium were interpreted in terms of the orientational order and molecular conformations. The molecular dynamics simulations of the dodecasaccharide were performed with explicit water and counterions, which are important factors that strongly influence molecular conformation. Subsequently, the results from computer simulation were used to generate a three-dimensional structure of the cyclic ECA which is consistent with the experimental NMR parameters.

Antigens, Bacterial↗
PubMed2001

Structure of a D-glycero -D-manno-heptan from the lipopolysaccharide of Helicobacter pylori.

A lipopolysaccharide (LPS) was isolated by hot phenol-water extraction from Helicobacter pylori strain D4 and found to contain no fucosylated poly-N-acetyllactosamine chain typical of most H. pylori strains studied but a homopolymer of D-glycero-D-manno-heptose (DD-Hep). The heptan attached to a core oligosaccharide was released by mild acid degradation of the LPS, and the following structure of the trisaccharide-repeating unit was established by chemical methods and 1H and 13C NMR spectroscopy: --> 2)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 -->. 1H NMR spectroscopy performed on small amounts of the intact LPS revealed the presence of the same polysaccharide in LPS of H. pylori strains D2 and D5, but not strain D10.

Helicobacter pylori↗
PubMed2001

Conformation of the glycosidic linkage in a disaccharide investigated by double-quantum solid-state NMR.

Double-quantum heteronuclear local field NMR is performed on a sample of a 13C2-labeled disaccharide, in which the two 13C spins are located on opposite sides of the glycosidic linkage. The evolution of the double-quantum coherences is found to be consistent with the solid-state conformation of the molecule, as previously determined by X-ray diffraction. The dependence of the double-quantum evolution on the glycosidic torsional angles is examined by using a graphical molecular manipulation program interfaced to a numerical spin simulation module.

Carbohydrate Conformation↗
PubMed2001

Molecular dynamics simulations of alpha-D-Manp-(1-->3)-beta-D-Glcp-OMe in methanol and in dimethyl sulfoxide solutions.

Molecular dynamics simulations have been performed of the disaccharide alpha-D-Manp-(1-->3)-beta-D-Glcp-OMe in two different solvents, namely in methanol and in dimethyl sulfoxide. The conformation of the disaccharide is similar to that previously determined in water. The three-dimensional structure around the solute was investigated by geometric hydrogen bonding criteria, radial distribution functions, coordination number analysis, residence times for hydrogen bonds, and spatial distribution functions. Differences and similarities between methanol and the aprotic dimethyl sulfoxide as solvent are analyzed.

Carbohydrate Conformation↗
PubMed2001

Structural analysis of the O-antigen polysaccharide from the Shiga toxin-producing Escherichia coli O172.

The structure of the O-antigen polysaccharide from Escherichia coli O172 has been determined. In combination with sugar analysis, NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by mass spectrometry and two-dimensional NMR techniques. An O-acetyl group was present as 0.7 equivalent per repeating unit. Treatment of the O-deacetylated polysaccharide with aqueous 48% hydrofluoric acid rendered cleavage of the phosphodiester in the backbone of the polymer and the pentasaccharide isolated after gel permeation chromatography was structurally characterized. Subsequent NMR experiments on polymeric materials revealed the structure of the repeating unit of the O-polysaccharide from E. coli O172 as:-->P-4)-alpha-D-Glcp-(1-->3)-alpha-L-FucpNAc-(1-->3)-alpha-D- GlcpNAc-(1-->3)-alpha-L-FucpNAc-(1-->4)-alpha-D-Glcp6Ac-(1-->

Carbohydrate Sequence↗
PubMed2000

Structure of a viscous exopolysaccharide produced by Lactobacillus helveticus K16.

A viscous extracellular polysaccharide produced by Lactobacillus helveticus K16 has been investigated. Sugar and methylation analysis, 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a hexasaccharide repeating unit. The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments. The structure of the repeating unit of the exopolysaccharide from L. helveticus K16 is as follows: carbohydrate sequence [see text].

Carbohydrate Sequence↗
PubMed2000

Structural elucidation of the viscous exopolysaccharide produced by Lactobacillus helveticus Lb161.

A viscous extracellular polysaccharide produced by Lactobacillus helveticus Lb161 isolated from raw milk has been investigated. Sugar and methylation analysis, and 1H and 13C NMR spectroscopy revealed that the polysaccharide is composed of a heptasaccharide repeating unit. The sequence of sugar residues was determined by use of two-dimensional nuclear Overhauser effect spectroscopy and heteronuclear multiple bond connectivity experiments. The structure of the repeating unit of the exopolysaccharide from L. helveticus Lb161 is as follows: carbohydrate structure [see text]. The polysaccharide contains approximately 0.6 equivalents of O-acetyl group per repeating unit (not located).

Animals↗
PubMed2000

Molecular dynamics simulation and nuclear magnetic resonance studies of the terminal glucotriose unit found in the oligosaccharide of glycoprotein precursors.

The trisaccharide alpha-d-Glcp-(1 --> 2)-alpha-d-Glcp-(1 --> 3)-alpha-d-Glcp-OMe, a model for the terminal glucotriose in Glc(3)Man(9)GlcNAc(2) in glycoprotein precursors, has been investigated by computer simulations and NMR spectroscopy. Molecular dynamics simulations were performed for 1 ns in aqueous solution and 20 ns in vacuo using the CHARMM-based force fields PARM22 and CHEAT95. An additional Monte Carlo simulation with the HSEA force field was also carried out. Experimental NMR data in water solution was obtained from measurement of long-range (1)H,(13)C heteronuclear trans-glycosidic coupling constants, (3)J(H,C), using one-dimensional Hadamard spectroscopy. Calculation of the (3)J(H,C) values from the simulations showed a varying degree of agreement to experimental data. It could be shown from simulation that the φ torsion angles differed, which was corroborated by the NMR measurements. Analyses were done of radial distribution functions and of hydrogen bonds. It was suggested that intermolecular hydrogen bonds were present, but in contrast to simulation the results from NMR spectroscopy did not support any major contribution. Hence, their influence on the conformation of the trisaccharide is rather small. Comparison of (1)H NMR chemical shifts for the trisaccharide and the glucotriose in Glc(3)Man(8)GlcNAc revealed high similarity. However, the derived conformation of the model substance in this work differed at one glycosidic torsion angle compared to the glucotriose on a large oligosaccharide.

Carbohydrate Conformation↗
PubMed2000

Structure of an atypical O-antigen polysaccharide of Helicobacter pylori containing a novel monosaccharide 3-C-methyl-D-mannose.

Lipopolysaccharides (LPS) were isolated by hot phenol-water extraction from Danish Helicobacter pylori strains D1, D3, and D6, which were nontypeable using a variety of anti-Lewis and anti-blood-group monoclonal antibodies. An atypical O-chain polysaccharide (PS) was liberated from the LPS of the three strains by acid under mild conditions and found to contain D-rhamnose (D-Rha), L-rhamnose (L-Rha), and a branched sugar, 3-C-methyl-D-mannose (D-Man3CMe). The last sugar, which has not hitherto been found in Nature, was identified using GLC-MS of the derived alditol acetate and the partially methylated alditol acetate, and (1)H and (13)C NMR spectroscopy, including NOESY and (1)H,(13)C HMBC experiments. The following structure of the trisaccharide repeating unit of the PS was established: -->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D- Rhap-(1-- >. In contrast to the pathogenic importance of the Lewis antigen mimicry exhibited by the PS of H. pylori strains previously investigated, the biological relevance of the atypical PS for H. pylori pathogenesis is unclear. The production of a differing surface PS may represent a form of antigenic variation by these particular H. pylori strains and/or may reflect the adaptation of these strains to a particular human population.

Acids↗
PubMed2000

Structure of the O-specific polysaccharide of Proteus mirabilis O11, another Proteus O-antigen containing an amide of D-galacturonic acid with L-threonine.

The O-specific polysaccharide of Proteus mirabilis O11 was studied by sugar analysis, Smith degradation, 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and 1H-detected 1H, 13C HMQC experiments. The following structure of a pentasaccharide repeating unit of the polysaccharide was established: [formual: see text] where D-GalA6LThr is N-(D-galacturonoyl)-L-threonine. ELISA with anti-P. mirabilis O11 serum showed that D-GalA6LThr is of minor importance for manifesting the O11 immunospecificity.

Amides↗