New Strategy for the Conformational Analysis of Carbohydrates Based on NOE and 13 C NMR Coupling Constants. Application to the Flexible Polysaccharide of Streptococcus mitis J22 † (original) (raw)
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Biochemistry, 1996
A method for constructing conformational models of flexible complex polysaccharides on the basis of NMR data and molecular modeling is described and is applied to a polysaccharide which is a lectin-binding receptor important in coaggregation of oral bacteria. The method involves uniform biosynthetic enrichment of the polysaccharide with 13 C which allows accurate measurements of heteronuclear coupling constants from a three-dimensional coupled HMQC-NOESY spectrum. The improved resolution of the 3D spectrum also provides a large number of accurate values of NOE cross peak volumes in a decoupled HMQC-NOESY spectrum. While it was not possible to construct a model for the flexible polysaccharide directly from the NOE data, a model was successfully built from the coupling constant data. Possible values of glycosidic dihedral angles were extracted from the 3 J CH data to build models which were evaluated by molecular modeling calculations. A simple average over a linear combination of low-energy conformations was selected which matched the experimental 3 J CH data within experimental error. Simulation of the NOE data for this same combination of conformers gave excellent agreement with experimental NOESY data. Molecular dynamics trajectories both with and without coupling constant constraints do not represent the experimental NOE and 3 J CH data as well as the linear combination model. While the polysaccharide has some flexibility in the antigenic site, the lectin-binding site, which contains a furanoside with (1f6)-linkages, provides a more flexible hinge in the polysaccharide.
Comparison of NMR and molecular modeling results for a rigid and a flexible oligosaccharide
Glycobiology, 1996
Three-bond heteronuclear coupling constants (3 J«/) are extremely useful in describing flexible models for oligosaccharides. We show that antiphase methods for measuring \JCH in oligosaccharides have limited reliability but that the coupling constants can be reliably measured in natural abundance by quantitative J-correlation methods. Interpretation of } i C H data for a pentasaccharide (lacto-N-fucopentaose 2) from human milk are consistent with a rigid model for the Lewis' trisaccharide epitope but for an antigenic tetrasaccharide fragment from the cell wall polysaccharide of viridans streptococci, 3 J C // data imply a considerably more flexible model. Nuclear Overhauser effect (NOE) data are reported for a heptasaccharide repeating unit isolated from the cell wall polysaccharide of Streptococcus gordonii 38. The results for a tetrasaccharide fragment are similar to data reported for the same fragment in the cell wall polysaccharide from S.mitis 322. This result implies a similar conformation for the tetrasaccharide fragment in the polysaccharide and in the heptasaccharide and also implies that anisotropy of motion is not significant in the interpretation of the nuclear Overhauser effects in the polysaccharide. Interpretation of the NOE results for the tetrasaccharide fragment, like the 3 J C // data, implies a flexible model with three conformations in fast exchange. The results of the two experimental techniques are combined with molecular modeling results including molecular dynamics simulation to provide a clear delineation between flexible and rigid oligosaccharide epitopes. The blood group Lewis" trisaccharide antigenic determinant is highly restricted in its motions by steric interactions while the antigenic tetrasaccharide fragment of the S.gordonii 38 heptasaccharide is considerably more mobile. We propose that some branched oligosaccharides are relatively rigid and some are flexible depending on subtle details of the linkages.
Physical chemistry chemical physics : PCCP, 2016
The conformation of saccharides in solution is challenging to characterize in the context of a single well-defined three-dimensional structure. Instead, they are better represented by an ensemble of conformations associated with their structural diversity and flexibility. In this study, we delineate the conformational heterogeneity of five trisaccharides via a combination of experimental and computational techniques. Experimental NMR measurements target conformationally sensitive parameters, including J couplings and effective distances around the glycosidic linkages, while the computational simulations apply the well-calibrated additive CHARMM carbohydrate force field in combination with efficient enhanced sampling molecular dynamics simulation methods. Analysis of conformational heterogeneity is performed based on sampling of discreet states as defined by dihedral angles, on root-mean-square differences of Cartesian coordinates and on the extent of volume sampled. Conformational c...
Biopolymers, 1996
We report on the conformation ofa tetrasaccharidefragment in the repeating subunit of the cell wall polysaccharide ofStreptococcus mitis 522, a receptor for the lectin ofActinomyces viscosus T14V in a bacterial coaggregation that is important in the ecological interactions oforal bacteria. Although there is considerable overlap of the 'H-nmr signals, some cross peaks can be extractedfrom conventional two-dimensional nuclear Overhauser effect spectroscopy (NOESY) data on the polysaccharide. These data cannot befit to a single conformation of the tetrasaccharide fragment. Therefore we have prepared a polysaccharide sample.fully enriched in 'jC from which we have determined accurate NOESY cross-peak volumes in a three-dimensional heteronuclear-resolved spectrum that allows accurate determination of many more NOESY cross peaks than does conventional two-dimensional spectroscopy. We have also used the "C enriched polysaccharide to measure accurate values of long-range l3C-' H coupling constants that can be correlated with glycosidic dihedral angles. Molecular modeling calcutations on the
Molecular conformations of a disaccharide investigated using NMR spectroscopy
Journal of Biomolecular NMR, 2006
The molecular structure of a-L-Rhap-(1 fi 2)-a-L-Rhap-OMe has been investigated using conformation sensitive NMR parameters: cross-relaxation rates, scalar 3 J CH couplings and residual dipolar couplings obtained in a dilute liquid crystalline phase. The order matrices of the two sugar residues are different, which indicates that the molecule cannot exist in a single conformation. The conformational distribution function, Pð/; wÞ, related to the two glycosidic linkage torsion angles / and w was constructed using the APME method, valid in the low orientational order limit. The APME approach is based on the additive potential (AP) and maximum entropy (ME) models. The analyses of the trajectories generated in molecular dynamics and Langevin dynamics (LD) computer simulations gave support to the distribution functions constructed from the experimental NMR parameters. It is shown that at least two conformational regions are populated on the Ramachandran map and that these regions exhibit very different molecular order.
Journal of Molecular Structure: THEOCHEM, 1997
Computational methods are intensively applied to explore the conformational space of oligosaccharides. In this report, the current modelling protocols for the conformational analysis of carbohydrates are reviewed. Approaches which need direct input of experimental data from NMR experiments (distance mapping and restrained molecular dynamics), various systematic search strategies (uniform rotation, relaxed and adiabatic maps) and random sampling techniques (random molecular mechanics calculations and Metropolis Monte Carlo simulations) are compared. Recently, molecular dynamics simulations have begun to play a dominant role for the conformational analysis of oligosaccharides. Therefore, various commonly used molecular dynamics simulation protocols (e.g. the use of different dielectric constants, simulated annealing and high-temperature simulation protocols, inclusion of explicit solvent molecules, the choice of appropriate non-bonded interaction cutoffs) which are increasingly used to obtain information about all the accessible conformations are discussed in detail. 0 1997 Elsevier Science B.V.
Biopolymers, 2000
A comparison of the conformation and dynamics of the cell wall polysaccharide of S. mitis J22 and the heptasaccharide repeating unit made from this polysaccharide was performed on the basis on nmr data. We have previously reported a model for this highly flexible polysaccharide in which four residues of the antigenic epitope adopt a defined conformation as do the two residues of the lectin-binding epitope. These domains are connected by a 6-substituted galactofuranoside residue that acts as a flexible hinge and the repeating subunits are joined by phosphodiester linkages that provide further flexibility. Homonuclear nuclear Overhauser effect (NOE) and longrange CC and C-H scalar coupling constants measured in uniform 13 C-labeled samples of the polysaccharide and heptasaccharide were very similar, indicating a similar conformational average in solution. Significant differences in the solution dynamics were found from the heteronuclear relaxation data, T 1 , T 1 , and NOE, which reflect the faster molecular tumbling of the heptasaccharide. Internal motions occurring on a picosecond time scale are relatively uniform along the polymer while dynamics on the time scale longer than a few nanoseconds is characteristic of hinge motion.
Chembiochem : a European journal of chemical biology, 2017
Exploration of the conformational spaces of flexible oligosaccharides is essential to gain deeper insights into their functional mechanisms. Here we characterised dynamic conformation of a high-mannose-type dodecasaccharide with a terminal glucose residue, a critical determinant recognised by molecular chaperones. The dodecasaccharide was prepared by our developed chemoenzymatic technique, which uses (13) C labelling and lanthanide tagging to detect conformation-dependent paramagnetic effects by NMR spectroscopy. The NMR-validated molecular dynamics simulation produced the dynamic conformational ensemble of the dodecasaccharide. This determined its spatial distribution as well as the glycosidic linkage conformation of the terminal glucose determinant. Moreover, comparison of our results with previously reported crystallographic data indicates that the chaperone binding to its target oligosaccharides involves an induced-fit mechanism.