Molecular basis of C(2+)-induced gelation in alginates and pectins: the egg-box model revisited.

For many ionic polysaccharides, the ability to form gels in the presence of divalent cations such as calcium is the key to biological functions and technological applications. This is particularly true for alginates and pectins, where the regular occurrence of respectively alpha-L-(1-4)-guluronate residues and alpha-D-galacturonate residues generates ordered templates for polymer chain associations that are involved in physical gels. The molecular basis responsible for the strength and the stereospecificity of calcium interactions for the two polysaccharides were investigated in a previous paper (Braccini; et al. Carbohydr. Res. 1999, 119). In the present work, a novel molecular modeling procedure has been developed; it involves a pairing procedure that evaluates all the possible associations of the ordered polyuronate chains with calcium ions to form dimers. Starting from the stable ordered forms of polygalacturonate and polyguluronate, all possible ways to form Ca(2+)-bridged dimers were computed; the parallel and antiparallel relative arrangements of the chains were also considered. Despite the structural analogy between polyguluronate and polygalacturonate chains, significant differences at the level of chain-chain associations are found. The popular "egg box model" can still be referred to in the case of polyguluronate. However, it cannot be used to describe a pectate junction zone as the unique feature of two consecutive chelation site per repeat, that provides a favorable entropic contribution to the interchain association is not reproduced by this pioneering model. The body of these results corroborates the two-stage process in the mechanism of calcium gelation, where the formation of strongly linked dimer associations is followed by the formation of weak inter-dimer associations mainly governed by electrostatic interactions.

Conformational behavior of hyaluronan in relation to its physical properties as probed by molecular modeling.

Hyaluronan (HA) is a linear charged polysaccharide whose structure is made up of repeating disaccharide units. Apparently conflicting reports have been published about the nature of the helical structure of HA in the solid state. Recent developments in the field of molecular modeling of polysaccharides offer new opportunities to reexamine the structural basis underlying the formation and stabilization of ordered structures and their interactions with counterions. The conformational spaces available and the low energy conformations for the disaccharide, trisaccharide, and tetrasaccharide segments of HA were investigated via molecular mechanics calculations using the MM3 force field. First, the results were used to access the configurational statistics of the corresponding polysaccharide. A disordered chain having a persistence length of 75 A at 25 degrees C is predicted. Then, the exploration of the stable ordered forms of HA led to numerous helical conformations, both left- and right-handed, having comparable energies. Several of these conformations correspond to the experimentally observed ones and illustrate the versatility of the polysaccharide. The double stranded helical forms have also been explored and theoretical structures have been compared to experimentally derived ones.

Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: a molecular modeling investigation.

Modeling simulations have been performed on the four regular glycuronans: alpha-D-(1--->4) polygalacturonic, alpha-L-(1--->4) polyguluronic, beta-D-(1--->4) polymannuronic, and beta-D-(1--->4) polyglucuronic acids. The goal of this study was to characterize the similarities and differences in conformational and configurational behavior as well as in calcium binding in order to progress in the understanding of the physicochemical properties of the parent polysaccharides of industrial interest, namely pectin, alginate and glucuronan. This required the evaluation of the accessible conformational space for the disaccharide subunits of the four homopolymers, using the flexible residue protocol of the MM3 molecular mechanics procedure. The results were used to access the configurational statistics of representative polysaccharide chains, as well as for the determination of the regular polysaccharide helices and their conformational transitions. The surfaces of all regular helices likely to occur for each polyuronide were explored for cation binding using the GRID procedure. Both alpha-D-(1--->4) polygalacturonate and alpha-L-(1--->4) polyguluronate chains exhibit a high specificity for calcium binding, and have well-defined chelation sites. In contrast, beta-D-(1--->4) polymannuronate and beta-D-(1--->4) polyglucuronate chains do not display any stereospecificity for calcium binding. The results gathered from molecular modeling lead to a clear understanding of the different structural features that are displayed by the four ionic polymers.

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

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 < tau(c) < 5 ns) show that rates of internal motion can be fairly precisely defined with respect to overall tumbling. Experimental data for a variety of oligosaccharides clearly indicate similar timescales for internal and overall motion.

Travelling on the potential energy surfaces of carbohydrates: comparative application of an exhaustive systematic conformational search with an heuristic search.

The calculated ensembles found by a heuristic conformational search algorithm, CICADA, for three small carbohydrates, ethyl beta-lactoside, methyl alpha-D-galactoside, and methyl beta-D-galactoside, are evaluated in terms of their ability to reproduce time-averaged optical rotation and NMR data. A unique dynamic model for methyl beta-D-galactoside has been obtained by fitting experimental NOESY volumes to the theoretical ones elaborated from the CICADA ensemble internuclear distances with the model-free formalism. In the case of ethyl beta-lactoside, the CICADA ensemble is compared to that of an exhaustive systematic grid-search method. The CICADA algorithm proved to be a very efficient method to find most of the important minima on even very complex potential energy surfaces, and the spectral quality of the CICADA ensemble was found to be of equal quality, if not superior, to that of the exhaustive systematic grid-search method. The CICADA algorithm has several advantages over other conformational search algorithms: (1) It has polynomial dependence of dimensions on computer time in contrast to the grid search, which has exponential dependence, (2) the conformations found are free of artificial harmonic constraint potentials, (3) it passes all barriers amongst families of conformations on conformational hypersurface but spends almost all its time in the essential highly populated areas, (4) the inherent properties of the algorithm make rigorous minimization criteria superfluous and provide good convergence behavior, and (5) as an important spin-off, it provides low-energy interconversion pathways that can, amongst others, be used for estimating adiabatic rotational barriers.

Structural analysis of cyclamen seed xyloglucan oligosaccharides using cellulase digestion and spectroscopic methods.

Xyloglucan polymers have been isolated from cyclamen seeds and characterized by both liquid and CP-MAS 13C NMR spectroscopy. Treatment of the polysaccharides with cellulase from Trichoderma viride afforded XG oligomers which have been studied with both mass spectrometry and NMR spectroscopy. The repeating unit in the intact polymers and the most abundant hydrolysis product correspond to the Gal1 Xyl3 Glc4 (XXLG) fragment. However, detection of notable amounts of Xyl3 Glc4 (XXXG) and Gal2 Xyl3 Glc4 (XLLG) indicates that the galactose distribution in xyloglucan from cyclamen is irregular. FAB-MS analysis of a new derivative, prepared by forming the glycosamine of m-tetrafluoroethoxy aniline, has led to unambiguous sequence information for the XXLG oligomer.

Conformational analysis of disaccharides using molecular dynamics and NMR methods.

The internal dynamics of four disaccharides, a galacturonic acid dimer, beta-ethyl-lactoside, sucralose and arabinobiose, have been studied using molecular dynamics and nuclear magnetic resonance methods. Theoretical motion models, which include order parameters and correlation times for internal motions, were extracted from dynamics trajectories in vacuo. In parallel, theoretical NOESY volumes were calculated from averaged distance matrices using 'model-free' spectral densities. These data were least-squares fitted to the normalized experimental NOESY volumes using the order parameters and internal motion correlation times as adjustable parameters. The resulting 'experimental' motional models were in good agreement with the theoretical ones with the exception of the galacturonic acid dimer which was best described by the rigid molecule approximation.

Internal motion in carbohydrates as probed by n.m.r. spectroscopy.

In the present study a combination of proton and carbon relaxation rates have been measured for several oligosaccharides at different temperatures. Correlation times, which have been calculated from both sets of data, have been compared in an attempt to establish the relative rate of internal motion. All the data suggest that these motions are not slow with respect to the overall tumbling.