Showing papers by "Serge Pérez published in 1997"

Structural basis of the properties of an industrially relevant thermophilic xylanase.

Abstract: A thermophilic xylanase from Bacillusstrain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60°C and relatively stable at high temperatures, with a temperature optimum of 75°C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of BacillusD3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or “sticky patches” between pairs of molecules. These “sticky patches” on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase. Proteins 29:77–86, 1997. © 1997 Wiley-Liss, Inc.

Internal motions and hydration of sucrose in a diluted water solution.

Abstract: This paper represents a synthesis of our most recent work on the hydration, internal and overall dynamics of sucrose in a diluted water solution. The studies were carried out as a total ensemble of 1.2 nanosecond condensed phase molecular dynamics trajectories. In this study the focus is on a 500 ps trajectory starting with the solute in the crystalline conformation. The presence of water was found to significantly alter the accessible conformational space of the solute. All potential intra-molecular hydrogen bonds were found to be exchanged to surrounding water molecules and the simulations suggest that the sucrose conformation is stabilized by the dynamic presence of two interring bridging water molecules: O-2g…Ow…O-3f and O-2g…Ow…O-lf. The overall shift in conformation of the solute induced by the presence of water was found to improve the theoretical models of experimental traits. It is demonstrated that the hydration structure and the internal and overall motions of sucrose compare extremely well with NMR data such as glycosidic heteronuclear coupling constants and the molecular tumbling time, with X-ray data of two partially hydrated sucrose structures in a protein complex and with translational diffusion coefficients and hydration numbers established from experimental studies. © 1997 by Elsevier Science Inc.

Transferred Nuclear Overhauser Enhancement (NOE) and Rotating‐Frame NOE Experiments Reflect the Size of the Bound Segment of the Forssman Pentasaccharide in the Binding Site of Dolichos Biflorus Lectin

Abstract: A complex between the Forssman pentasaccharide α-D-GalNAc-(13)-β-D-GalNAc-(13)-α-D-Gal-(14)-β-D-Gal-(14)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety α-D-GalNAc-(13)-β-D-GalNAc compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the non-reducing disaccharide moiety α-D-GalNAc-(13)-β-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion α-D-Gal-(14)-β-D-Gal-(14)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment α-D-GalNAc-(13)-β-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Validation of two conformational searching methods applied to sucrose : simulation of nmr and chiro-optical data

Abstract: The potential energy hypersurface of sucrose has been examined by molecular mechanics calculations (MM3(92)) interfaced with two different algorithms for conformational searching. The first uses the systematic grid-search approach. The second algorithm, which has a lower CPU requirement, is the so-called CICADA procedure (Channels In Conformational space Analyzed by Driver Approach). This method drives selected torsional angles to explore the low-energy regions and permits full geometry relaxation. Using the grid-search approach, the adiabatic relaxed map of sucrose was calculated as a function of the glycosidic torsion angles, and three families of stable conformers were identified. The CICADA procedure found all minima and also the low-energy conversion pathways for the disaccharide in agreement with those located by the grid-search approach. The method appears to be more efficient for determining the global energy minimum. Beside locating structures at energy minima, transition states and low-energy pathways were also identified. In order to assess the validity of the potential energy surfaces, comparisons were made between experimental and theoretical values of some spectral properties. Optical rotation values were taken from the literature, whereas the NOESY volumes were measured at 400 MHz. Theoretical data were calculated taking into account all the accessible conformations derived from either the grid-search or the CICADA method. NMR relaxation data were calculated using averaging methods appropriate for both slow and fast internal motions. The present study demonstrates the ability of the new CICADA method to reproduce the potential energy surface of a flexible molecule and therefore successfully to simulate its spectral properties.

Conformational analysis of biantennary glycans and molecular modeling of their complexes with lentil lectin.

Abstract: Some mannose-binding legume lectins show higher affinity for fucosylated glycans than for glycans without fucose. These lectins possess a secondary binding site. Owing to the possibility of additional fucose binding, oligosaccharides adopt different conformations depending on whether they contain fucose or not. To study these conformational differences, complexes of fucosylated and unfucosylated glycans with Lens culinaris lectin have been modeled. Starting points were X-ray structures of lentil lectin and complexes of the homologous Lathyrus ochrus lectin. The SYBYL molecular modeling package with the TRIPOS force field was used. Two different models were built, displaying in both a network of hydrogen bonds between the saccharide and the binding site. Furthermore, to compare the free and bound ligand, conformational analysis in the free state has been performed. A complete analysis of all possible disaccharide fragments has been performed using the MM3 force field. A CICADA analysis employing the same force field was carried out to study the complete oligosaccharide. Low-energy conformers found by CICADA were clustered in conformational families and analyzed in terms of flexibility and rotational barriers. All values of glycosidic torsion angles are in the range as calculated by MM3 for the disaccharides.

Modeling Polysaccharides: Present Status and Challenges

Abstract: The most recent tools that have been developed for modeling the three-dimensional features of polysaccharides and carbohydrate polymers are presented. The presentation starts with a description of the conformations of the monosaccharides, and of the flexible rings such as in the case of five-membered rings, and a thorough description of the conformational space that is available for a disaccharide unit, either in vacuo or in an aqueous phase. The extension to the modeling of the parent polysaccharides is addressed, based on the assumption that owing to the size and relative rigidity of the intervening monosaccharides units, the rotations at a particular linkage can be, under some conditions, considered as independent of nearest neighbor interactions. Appropriate modeling techniques are described that can provide insights into the dimensions of the chain in a solution which is best described as a random coil accompanied by the occurrence of local "helical" regions. With the help of such descriptors such as helical parameters, the ordered state of polysaccharide strands can be readily characterized. The generation of double or triple helices can be then attempted in order to explore the occurrence of such multi-stranded arrangements that may be energetically stable. The final step in the determination of the structure of polysaccharides in the ordered state, is the investigation of the interactions of different helices. This may lead to either the best arrangement(s) between two polymeric chains, or to the prediction of the dimensions, and the symmetry of a three-dimensional lattice. Some of the tools which have been developed should allow automatic scarches for meaningful correlations between structures and functions, through exploratory data analysis. Structure-function or structure-property correlation could be then used to model changes arising from structural alterations. This would open the field of polysaccharide engineering.

Stereochemical analysis of d-galacto-sulfoxides using (S)-α-methoxyphenylacetic acid

Abstract: ( S )- α -Methoxyphenylacetic acid (MPAA) was used as an NMR shift reagent to predict the absolute configuration of some sulfoxide-containing sugars. The correctness of this assignment was confirmed by X-ray crystallography.

Combined NMR, grid search/MM3 and Metropolis Monte Carlo/GEGOP studies of two l-fucose containing disaccharides: α-l-Fuc-(1,4)-β-d-GlcNAc-OMe and α-l-Fuc-(1,6)-β-d-GlcNAc-OMe

Abstract: Complete proton NMR data provided a firm experimental basis to infer the conformational properties of the Lewis type disaccharide α- l -Fuc-(1,4)-β- d -GlcNAc-OMe 1 and the N -glycoprotein type disaccharide α- l -Fuc-(1,6)-β- d -GlcNAc-OMe 2 in aqueous solution. Relaxed potential energy maps from systematic grid searches (GS) using the MM3 force field and Metropolis Monte Carlo (MMC) simulations employing the GEGOP force field were used to calculate corresponding ensemble average NMR data such as 1D transient NOE curves and vicinal coupling constants J ( H , H ). For the disaccharide with a flexible (1,6) linkage ( 2 ), the relaxed potential energy surface based on the MM3 force field was calculated with three variable dihedral angles, φ, ψ and ω. R -factors derived from a comparison of experimental and theoretical NOE data allowed an evaluation of the quality of the conformational models derived from the calculations. Seven inter glycosidic 1D transient NOE curves were measured for each of both disaccharides, 1 and 2 . Overall R -factors of approximately 17% for the 1–4 linked disaccharide and 20% for the 1–6 linked disaccharide indicate a very satisfying agreement between theoretical calculations, both GS/MM3 and MMC/GEGOP, and experimental NOE data, indicating that the gross conformational picture developed is realistic. On the other hand, a close inspection of individual NOE curves and vicinal coupling constants 3 J ( H 5, H 6-pro-R) and 3 J ( H 5, H 6-pro-S) with corresponding theoretical values revealed shortcomings of the computational methods applied. Firstly, the conformational equilibrium around the C5C6 bond of the GlcNAc unit in disaccharide 1 is not correctly described by the GS/MM3 method. This can lead to a false interpretation of NOE data involving protons attached to C6 of GlcNAc. Secondly, relaxation of ring geometry (GS/MM3) was found to have a measurable improvement of intra glycosidic NOEs in both disaccharides, 1 and 2 . In summary, the conformational models derived for disaccharides 1 and 2 represent a starting point for further analysis of potential conformational changes that may occur upon binding of these compounds to specific receptor proteins such as lectins or antibodies.

NMR analysis of carbohydrates with model-free spectral densities: the dispersion range revisited

Abstract: Over the past decade molecular mechanics and molecular dynamics studies have demonstrated considerable flexibility for carbohydrates. In order to interpret the corresponding NMR parameters, which correspond to a time-averaged or 'virtual' conformer, it is necessary to simulate the experimental data using the averaged geometrical representation obtained with molecular modelling methods. This structural information can be transformed into theoretical NMR data using empirical Karplus-type equations for the scalar coupling constants and the appropriate formalism for the relaxation parameters. In the case of relaxation data, the 'model-free' spectral densities have been widely used in order to account for the internal motions in sugars. Several studies have been conducted with truncated model-free spectral densities based on the assumption that internal motion is very fast with respect to overall tumbling. In this report we present experimental and theoretical evidence that suggests that this approach is not justified. Indeed, recent results show that even in the case of moderate-sized carbohydrates internal motions are occurring on the same timescale as molecular reorientation. Simulations of relaxation parameters (NOESY volumes, proton cross-relaxation rates, carbon T1 and nOe values) in the dispersion range (0.1