Structure and properties of a bacterial polysaccharide named Fucogel.

The chemical structure of a polysaccharide named Fucogel was characterized and the position of acetylation was identified by NMR. A conformational analysis was performed on this 3-sugar repeating unit. From this, the persistence length, characterizing the stiffness of the polysaccharide, was determined and the role of the presence of acetyl group, reducing the stiffness, was pointed out. The helical conformations were also predicted, one of these being in agreement with X-ray data obtained on a similar polysaccharide. Experimental characterization of the native and deacetylated polysaccharides was developed. SEC experiments allowed us to determine the molar mass and the persistence length on the deacetylated polysaccharide. The value is in good agreement with that predicted from the molecular modeling. Microcalorimetry, rheology, and fluorescence spectroscopy demonstrated respectively that no helical conformation exists in solution but that loose interchain interactions due to the acetyl substituents exist in dilute solutions.

A priori crystal structure prediction of native celluloses.

The packing of beta-1,4-glucopyranose chains has been modeled to further elaborate the molecular structures of native cellulose microfibrils. A chain pairing procedure was implemented that evaluates the optimal interchain distance and energy for all possible settings of the two chains. Starting with a rigid model of an isolated chain, its interaction with a second chain was studied at various helix-axis translations and mutual rotational orientations while keeping the chains at van der Waals separation. For each setting, the sum of the van der Waals and hydrogen-bonding energy was calculated. No energy minimization was performed during the initial screening, but the energy and interchain distances were mapped to a three-dimensional grid, with evaluation of parallel settings of the cellulose chains. The emergence of several energy minima suggests that parallel chains of cellulose can be paired in a variety of stable orientations. A further analysis considered all possible parallel arrangements occurring between a cellulose chain pair and a further cellulose chain. Among all the low-energy three-chain models, only a few of them yield closely packed three-dimensional arrangements. From these, unit-cell dimensions as well as lattice symmetry were derived; interestingly two of them correspond closely to the observed allomorphs of crystalline native cellulose. The most favorable structural models were then optimized using a minicrystal procedure in conjunction with the MM3 force field. The two best crystal lattice predictions were for a triclinic (P(1)) and a monoclinic (P2(1)) arrangement with unit cell dimensions a = 0.63, b = 0.69, c = 1.036 nm, alpha = 113.0, beta = 121.1, gamma = 76.0 degrees, and a = 0.87, b = 0.75, c = 1.036 nm, gamma = 94.1 degrees, respectively. They correspond closely to the respective lattice symmetry and unit-cell dimensions that have been reported for cellulose Ialpha and cellulose Ibeta allomorphs. The suitability of the modeling protocol is endorsed by the agreement between the predicted and experimental unit-cell dimensions. The results provide pertinent information toward the construction of macromolecular models of microfibrils.

A conformational study of the xyloglucan oligomer, XXXG, by NMR spectroscopy and molecular modeling.

A structural study of the XXXG xyloglucan heptasaccharide (X = alpha-D-Xylp(1 --> 6)-beta-D-Glcp and G = beta-D-Glcp) isolated from apple fruit has been undertaken with nmr and molecular mechanics methods. Quantitative 400 MHz nmr data including nuclear Overhauser effect spectroscopy (NOESY) volumes were recorded at both 6 and 20 degrees C. In spite of severe overlapping of resonances, it was possible to estimate summed NOEs for the majority of the anomeric and glucosyl methylene protons. An ensemble-average population of preferred geometries has been established with the CICADA conformational searching algorithm associated with the MM3 force field. Comparison of the theoretical data obtained by back-calculation of the NOESY volumes from the ensemble-average distance matrix program and motional models based on the Stokes-Einstein-Debye relation satisfactorily reproduce the experimental data. Conformational averaging about the mainchain glycosidic linkages includes both the syn and anti conformers and a minor gauche-gauche population is highly probable. The theoretical data overestimate the syn preference of the Glc(c) --> Glc(b) linkage as well as the Glc(c) GT rotamer population. Finally, both the motional models and the conformational search indicate a fairly rigid backbone and greater flexiblity for the xylose side chains.

The preferred conformations of the four oligomeric fragments of Rhamnogalacturonan II.

Rhamnogalacturonan II (RG-II) is a structurally complex pectic mega-oligosaccharide that is released enzymatically from the primary cell wall of higher plants. RG-II contains 28 monosaccharide units (MW approximately equal to 6 KDa) which belong to 12 different families of glycosyl residues, including very unusual ones such as Kdo, Dha, aceric acid, and apiose. Eighteen different disaccharide segments can be identified, and so far the primary structure has not yet been determined. These monomeric units are arranged into four structurally well-defined oligosaccharide side chains, linked to a pectic backbone made up of 1,4-linked alpha-D-galactosyluronic acid residues. The specific attachment sites of these four side-chains on the pectic backbone remains to be elucidated. The present work presents a three-dimensional database of all the monosaccharide and disaccharide components of RG-II. The conformational behavior of D-Apif and L-AceAf monosaccharide has been assessed through computations performed with the molecular mechanics program MM3 using the flexible residue approach. For each furanosyl residue, energies of various envelope and twist conformers were systematically calculated as a function of the puckering parameters Q and phi. Energy minima are observed in both the Northern and Southern zones of the conformational wheel of each monosaccharide. As for the constituting segments, the conformational behaviour of 18 different disaccharides was evaluated using the flexible residue procedure of the MM3 molecular mechanics procedure. For each disaccharide, the adiabatic energy surface, along with the locations of the local energy minima and drawings of the conformations of each local minimum located in the energy maps have been established. The geometries of the minima and the potential energy surfaces of the different fragments were included in the database of the POLYS, a program for building oligo and polysaccharides. All these results were used for the generation, prior to a complete optimization, of the complete structure of each fragment of RG-II. It is shown that both A and B fragments are very flexible about the two sidechain glycosidic linkages which are closest to the backbone. The remaining part of the sidechain is rigid for the heavily branched A fragment, it is flexible for the more linear B fragment. The lowest energy conformer of each fragment results in good exposure of the hydroxyl groups of the apiosyl residues. Some possible implications of these features in boron complexation are presented.

Conformational properties of a cycle oligosaccharide: cyclotrikis-(1-->6)-[alphaD-glucopyranosyl-(1-->4)-beta-D-glucopyranosy l].

The title compound is a cyclic oligosaccharide having six glucopyranose residues linked alternatively by alpha-(1-->4) and beta-(1-->6) glycosidic linkages. Like cyclodextrin analogues it is expected to exhibit an internal cavity and to form inclusion complexes with other species. In order to investigate its conformational preferences, an extensive conformational search was carried out using a combination of Metropolis Monte-Carlo (MMC) procedure in the glycosidic torsion angle space and molecular mechanics procedures. To this end a specific program (METROCYCLIX) was developed. To reduce the MMC search, conformational maps of parent disaccharides were considered as starting entries. Fully minimized conformations were gathered into families using a clustering technique based on RMS fitting over the glycosidic torsion angle values. A wide range of local energy minima were identified in spite of ring closure conditions that constrained the structure of the oligosaccharide. Low energy conformers were stabilized by intramolecular interactions between distant residues. From the Bolzmann population of the best structures derived from the clustering results, various average properties were calculated and compared with experimental data obtained by high resolution NMR. Interpretation of these experimental values (heteronuclear coupling constants, rotating frame nuclear Overhauser effects, relaxation times) relies on the use of Karplus like equations (coupling constants) and analysis of the full relaxation rate matrix treatment (ROE). The quality of the molecular modelling strategy used is assessed by the agreement obtained between calculated and measured observables.

Modeling polysaccharides: present status and challenges.

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.

Hydration of alpha-maltose and amylose: molecular modelling and thermodynamics study.

Hydration of alpha-maltose and amylose were investigated using molecular modelling and thermodynamics methods. The structure and energy of hydration of three low-energy conformers of alpha-maltose were determined by the MM3 molecular mechanics method. The hydration structure was found to be sensitive to the conformation of alpha-maltose and hydration numbers 10 or 11 were estimated for the different conformers. Differential scanning calorimetry and thermogravimetric analysis were used to determine the number of water molecules specifically bonded (non-freezing water) to amylose and different samples of alpha-maltose. Due to high crystallinity of alpha-maltose samples, the observed non-freezing water content was lower than predicted by molecular modelling. In contrast, the experimental number of non-freezing molecules of water per D-glucopyranose residue for amorphous amylose (nh = 3.8) is in good accordance with the value of 3.8 extracted from our calculations.

Conformational analysis of carbohydrates inferred from NMR spectroscopy and molecular modeling. Applications to the behavior of oligo-galactomannan chains.

In both the biological functions and industrial applications of carbohydrates, their conformational behavior along with the dynamic fluctuations that their structures experience are of great significance. The elucidation of the three-dimensional characteristics of carbohydrates and carbohydrate-containing molecules is in general performed by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Heteronuclear couplings and nuclear Overhauser effect (NOE) data are major tools for structural determination. The angular dependence of carbon-proton coupling constants has been shown to depend also on the pathways involved. Concerning molecular modeling, the goal is to generate the ensemble of structures that are consistent with the experimental data. This approach is illustrated in the galactomannan chain. It consists of the conformational study of two model disaccharides (mannobiose and epimelibiose) of galactomannans through carbon-proton coupling constants and NOE measurements. Experimental data are then compared to results of conformational analysis that predicted the stable conformers in solution. From these results helical conformations of oligomers are generated and are discussed in terms of the influence of the position and number of galactose units on the conformation of the mannan backbone.

Conformation and dynamics of a cyclic (1-->2)-beta-D-glucan.

A molecular modelling and nuclear magnetic resonance spectroscopic study was performed in order to gain insight into the conformational preferences of cyclosophoroheptadecaose. MM3 molecular mechanics calculations predicted a non-symmetric conformer with a small cavity of 3.7 A diameter as the lowest energy form. Molecular dynamics simulations gave insight into the dynamics of the free cyclosophoroheptadecaose and also supported the results of molecular mechanics calculations. A fair agreement was found between experimental data and corresponding average values predicted by molecular modelling.

Conformational study of digalacturonic acid and sodium digalacturonate in solution.

The solution conformation of digalacturonic acid and its sodium salt have been analyzed using nuclear magnetic resonance data and molecular mechanics calculations. The flexibility around the glycosidic linkage was characterized by calculation of the relaxed (phi, psi) potential surfaces for the isolated molecule, and also for dimethyl sulfoxide and aqueous solutions using the CHARMM and SOLVOL programs. The one-bond and three-bond proton-carbon couplings were measured and H-1'-H-4 distances were estimated from NOESY experiments. The calculated potential surfaces were used to determine theoretical ensemble averages of NMR data. The agreement between the experimental and theoretical data is very satisfactory. The calculations show a strong effect of solvent on the solution behavior of both compounds. The vacuum lowest energy conformer of digalacturonic acid is stabilized by solvation, while for sodium digalacturonate the solvent induces a conformational change. An extrapolation of the stable conformers to polysaccharide chains implies that poly(galacturonic acid) occurs in solution as a three-fold helix and sodium poly(galacturonate) as a two-fold helix.

PCILO quantum-mechanical relaxed conformational energy map of methyl 4-thio-alpha-maltoside in solution.

A theoretical study is presented of the conformational properties of methyl 4-thio-alpha-maltoside. Quantum-chemical PCILO (perturbed configuration interaction with localized orbitals) energy minimization with evaluation of the solvent effect has been used to calculate semi-rigid and relaxed (phi, psi) conformational-energy surfaces in 1,4-dioxane, methanol, dimethyl sulfoxide, and water. The inclusion of molecular flexibility in the conformational analysis of this disaccharide derivative was found to have a significant effect upon the allowed conformational space of the molecule. Calculations revealed the existence of 15 stable conformers having different internal geometries. The relative abundance of these conformers is strongly influenced by solvent. The locations of the global minima and "virtual" conformations vary with solvent and the flexibility of methyl 4-thio-alpha-maltoside significantly decreases from 1,4-dioxane to water. It was found that the conformational properties of methyl 4-thio-alpha-maltoside differ from those of the corresponding 4-oxygenated analogue, methyl alpha-maltoside. The calculated solution behaviour is supported by very good agreement between the theoretical values mean value of 3JC-4,H-1' = 2.67 and mean value of 3JC-1',H-4 = 5.24 Hz calculated as ensemble averages over all conformers on the relaxed map and the experimental values of 2.95 and 5.15 Hz in water solution.

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: I. A 1H and 31P n.m.r. study.

The structure of an intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex has been investigated by using ultraviolet absorption, circular dichroism, 1H and 31P n.m.r. The binding of cis-DDP does not inhibit the formation of a duplex but it induces a lowering of congruent to 26 degrees C of its melting temperature. A broadening of the 1H spectrum prevents an accurate analysis of the platination site. Nevertheless, by considering its thermal behavior and the number of imino protons a model of structure of the platinated duplex is proposed in which the central C.G. pair is disrupted and a neighboring C.G pair is very accessible or distorted. The environment of two phosphate groups is disturbed by the cis-DDP binding.

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: II. A molecular mechanics modeling study.

Molecular mechanics modeling has been carried out for the intrastrand cis-[Pt(NH3)2(d(pGpCpG))] adduct in a dodecanucleotide duplex. In a first step, an analysis of the conformation of a platinated trinucleotide shows the tendency of the two chelating guanines to take respectively anti-syn or syn-anti positions and the role of phosphate-platinum amino group interactions. In a second step, duplex structures are examined. The distortions induced by platination are especially analysed for kinked and unkinked forms of the dodecanucleotide. In all models the central C.G pair in the platination site is disrupted and the double helix is more disturbed on the 5' side of this site than on the 3' side. Most of the structural features arising from the modeling are in agreement with the conclusions of an n.m.r. study reported in the preceding paper.

Two-dimensional 1H NMR study of d(br5C-G)3 in the Z-form. Self association and flexibility of the left-handed double helix.

The Z conformation of the auto complementary hexanucleoside pentaphosphate d(br5C-G)3 in 1 M NaClO4 solution has been investigated by using 2D NMR techniques. NOESY experiments performed at different temperatures show that the oligonucleotide exhibits end-to-end associations at room temperature. The conformation of the hexanucleotide molecules is very similar to that found in the crystal which was described by Chevrier et al. (J. Mol. Biol., 1986, 188, 707-719) as a Z-I form. When the temperature is increased the aggregates are dissociated and a conformational change is observed which is interpreted as a Z-I in equilibrium Z-II transition.