Delineating the conformational flexibility of trisaccharides from NMR spectroscopy experiments and computer simulations.
ABSTRACT: 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 clustering, based on the glycosidic linkage dihedral angles, shows that accounting for the full range of sampled conformations is required to reproduce the experimental data, emphasizing the utility of the molecular simulations in obtaining an atomic detailed description of the conformational properties of the saccharides. Results show the presence of differential conformational preferences as a function of primary sequence and glycosidic linkage types. Significant differences in conformational ensembles associated with the anomeric configuration of a single glycosidic linkage reinforce the impact of such changes on the conformational properties of carbohydrates. The present structural insights of the studied trisaccharides represent a foundation for understanding the range of conformations adopted in larger oligosaccharides and how these molecules encode their conformational heterogeneity into the monosaccharide sequence.
Project description:We present an extension of the all-atom internal-coordinate force field, ICMFF, that allows for simulation of heterogeneous systems including hexopyranose saccharides and glycan chains in addition to proteins. A library of standard glycan geometries containing α- and β-anomers of the most common hexapyranoses, i.e., d-galactose, d-glucose, d-mannose, d-xylose, l-fucose, N-acetylglucosamine, N-acetylgalactosamine, sialic, and glucuronic acids, is created based on the analysis of the saccharide structures reported in the Cambridge Structural Database. The new force field parameters include molecular electrostatic potential-derived partial atomic charges and the torsional parameters derived from quantum mechanical data for a collection of minimal molecular fragments and related molecules. The ϕ/ψ torsional parameters for different types of glycosidic linkages are developed using model compounds containing the key atoms in the full carbohydrates, i.e., glycosidic-linked tetrahydropyran-cyclohexane dimers. Target data for parameter optimization include two-dimensional energy surfaces corresponding to the ϕ/ψ glycosidic dihedral angles in the disaccharide analogues, as determined by quantum mechanical MP2/6-31G** single-point energies on HF/6-31G** optimized structures. To achieve better agreement with the observed geometries of glycosidic linkages, the bond angles at the O-linkage atoms are added to the internal variable set and the corresponding bond bending energy term is parametrized using quantum mechanical data. The resulting force field is validated on glycan chains of 1-12 residues from a set of high-resolution X-ray glycoprotein structures based on heavy atom root-mean-square deviations of the lowest-energy glycan conformations generated by the biased probability Monte Carlo (BPMC) molecular mechanics simulations from the native structures. The appropriate BPMC distributions for monosaccharide-monosaccharide and protein-glycan linkages are derived from the extensive analysis of conformational properties of glycoprotein structures reported in the Protein Data Bank. Use of the BPMC search leads to significant improvements in sampling efficiency for glycan simulations. Moreover, good agreement with the X-ray glycoprotein structures is achieved for all glycan chain lengths. Thus, average/median RMSDs are 0.81/0.68 Å for one-residue glycans and 1.32/1.47 Å for three-residue glycans. RMSD from the native structure for the lowest-energy conformation of the 12-residue glycan chain (PDB ID 3og2) is 1.53 Å. Additionally, results obtained for free short oligosaccharides using the new force field are in line with the available experimental data, i.e., the most populated conformations in solution are predicted to be the lowest energy ones. The newly developed parameters allow for the accurate modeling of linear and branched hexopyranose glycosides in heterogeneous systems.
Project description:Knowledge of the three-dimensional structures of glycans and glycoproteins is useful for a full understanding of molecular processes in which glycans are involved, such as antigen-recognition and virus infection, to name a few. Among the ubiquitous nuclei in glycan molecules, the (13)C nucleus is an attractive candidate for computation of theoretical chemical shifts at the quantum chemical level of theory to validate and determine glycan structures. For this purpose, it is important to determine, first, which carbons can be used as probes to sense conformational changes and, second, all factors that affect the computation of the shielding, at the density functional theory (DFT) level of theory, of those carbons. To answer such questions, we performed a series of analyses on low-energy conformations, obtained by sampling the glycosidic torsional angles (?, ?) every 10°, of 12 disaccharides. Our results provide evidence that: (i) the carbons that participate in the glycosidic linkage are the most sensitive probes with which to sense conformational changes of disaccharides; (ii) the rotation of the hydroxyl groups closest to the glycosidic linkage significantly affects the computation of the shieldings of the carbons that participate in the glycosidic linkage; (iii) it is not possible to obtain the shieldings of one disaccharide from the computed values of a different disaccharide or from those disaccharides that differ in the anomeric state; and (iv) a proper basis set distribution, a functional, and a step size, with which to sample the conformational space, are necessary to compute shieldings accurately and rapidly.
Project description:Computational models can provide detailed information about molecular conformations and interactions in solution, which is currently inaccessible by other means in many cases. Here we describe an efficient and precise coarse-grained model for long polysaccharides in aqueous solution at different physico-chemical conditions such as pH and ionic strength. The Model is carefully constructed based on all-atom simulations of small saccharides and metadynamics sampling of the dihedral angles in the glycosidic links, which represent the most flexible degrees of freedom of the polysaccharides. The model is validated against experimental data for Chitosan molecules in solution with various degree of deacetylation, and is shown to closely reproduce the available experimental data. For long polymers, subtle differences of the free energy maps of the glycosidic links are found to significantly affect the measurable polymer properties. Therefore, for titratable monomers the free energy maps of the corresponding links are updated according to the current charge of the monomers. We then characterize the microscopic and mesoscopic structural properties of large chitosan polysaccharides in solution for a wide range of solvent pH and ionic strength, and investigate the effect of polymer length and degree and pattern of deacetylation on the polymer properties.
Project description:A fermented beverage of plant extracts was prepared from about fifty kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). We have previously examined the preparation of novel four trisaccharides from the beverage: O-beta-D-fructopyranosyl-(2->6)-O-beta-D-glucopyranosyl-(1->3)-D-glucopyranose, O-beta-D-fructopyranosyl-(2->6)-O-[beta-D-glucopyranosyl-(1->3)]-D-glucopyranose, O-beta-D-glucopyranosyl-(1->1)-O-beta-D-fructofuranosyl-(2<->1)-alpha-D-glucopyranoside and O-beta-D-galactopyranosyl-(1->1)-O-beta-D-fructofuranosyl-(2<->1)- alpha-D-glucopyranoside.Three further novel oligosaccharides have been found from this beverage and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structural confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements.The following novel trisaccharides were identified: O-beta-D-fructofuranosyl-(2->1)-O-[beta-D-glucopyranosyl-(1->3)]-beta-D-glucopyranoside (named "3G-beta-D-glucopyranosyl beta, beta-isosucrose"), O-beta-D-glucopyranosyl-(1->2)-O-[beta-D-glucopyranosyl-(1->4)]-D-glucopyranose (4(1)-beta-D-glucopyranosyl sophorose) and O-beta-D-fructofuranosyl-(2->6)-O-beta-D-glucopyranosyl-(1->3)-D-glucopyranose (6(2)-beta-D-fructofuranosyl laminaribiose).
Project description:Twelve disaccharides containing β-(1→4) linkages and displaying systematic structural variations in the vicinity of these linkages were selectively labeled with 13C to facilitate measurements of multiple NMR spin-spin (scalar; J) coupling constants (JCH and JCC values) across their O-glycosidic linkages. Ensembles of spin-couplings (2JCOC, 3JCOCH, 3JCOCC) sensitive to the two linkage torsion angles, phi (ϕ) and psi (ψ), were analyzed by using parametrized equations obtained from density functional theory (DFT) calculations, Fredholm theory, and circular statistics to calculate experiment-based rotamer populations for ϕ and ψ in each disaccharide. With the statistical program MA'AT, torsion angles ϕ and ψ were modeled as a single von Mises distribution, which yielded two parameters, the mean position and the circular standard deviation (CSD) for each angle. The NMR-derived rotamer populations were compared to those obtained from 1 μs aqueous molecular dynamics (MD) simulations and crystallographic database statistical analyses. Conformer populations obtained exclusively from the MA'AT treatment of redundant J-couplings were in very good agreement with those obtained from the MD simulations, providing evidence that conformational populations can be determined by NMR for mobile molecular elements such as O-glycosidic linkages with minimal input from theory. The approach also provides an experimental means to validate the conformational preferences predicted from MD simulations. The conformational behaviors of ϕ in the 12 disaccharides were very similar, but those of ψ varied significantly, allowing a classification of the 12 disaccharides based on preferred linkage conformation in solution.
Project description:Glycosaminoglycans (GAGs) are the linear carbohydrate components of proteoglycans (PGs) and are key mediators in the bioactivity of PGs in animal tissue. GAGs are heterogeneous, conformationally complex, and polydisperse, containing up to 200 monosaccharide units. These complexities make studying GAG conformation a challenge for existing experimental and computational methods. We previously described an algorithm we developed that applies conformational parameters (i.e., all bond lengths, bond angles, and dihedral angles) from molecular dynamics (MD) simulations of nonsulfated chondroitin GAG 20-mers to construct 3-D atomic-resolution models of nonsulfated chondroitin GAGs of arbitrary length. In the current study, we applied our algorithm to other GAGs, including hyaluronan and nonsulfated forms of dermatan, keratan, and heparan and expanded our database of MD-generated GAG conformations. Here, we show that individual glycosidic linkages and monosaccharide rings in 10- and 20-mers of hyaluronan and nonsulfated dermatan, keratan, and heparan behave randomly and independently in MD simulation and, therefore, using a database of MD-generated 20-mer conformations, that our algorithm can construct conformational ensembles of 10- and 20-mers of various GAG types that accurately represent the backbone flexibility seen in MD simulations. Furthermore, our algorithm efficiently constructs conformational ensembles of GAG 200-mers that we would reasonably expect from MD simulations.
Project description:We present an extension of the CHARMM hexopyranose monosaccharide additive all-atom force field to enable modeling of glycosidic-linked hexopyranose polysaccharides. The new force field parameters encompass 1?1, 1?2, 1?3, 1?4, and 1?6 hexopyranose glycosidic linkages, as well as O-methylation at the C(1) anomeric carbon, and are developed to be consistent with the CHARMM all-atom biomolecular force fields for proteins, nucleic acids, and lipids. The parameters are developed in a hierarchical fashion using model compounds containing the key atoms in the full carbohydrates, in particular O-methyl-tetrahydropyran and glycosidic-linked dimers consisting of two molecules of tetrahyropyran or one of tetrahydropyran and one of cyclohexane. Target data for parameter optimization include full two-dimensional energy surfaces defined by the ?/? glycosidic dihedral angles in the disaccharide analogs as determined by quantum mechanical MP2/cc-pVTZ single point energies on MP2/6-31G(d) optimized structures (MP2/cc-pVTZ//MP2/6-31G(d)). In order to achieve balanced, transferable dihedral parameters for the ?/? glycosidic dihedral angles, surfaces for all possible chiralities at the ring carbon atoms involved in the glycosidic linkages are considered, resulting in over 5000 MP2/cc-pVTZ//MP2/6-31G(d) conformational energies. Also included as target data are vibrational frequencies, pair interaction energies and distances with water molecules, and intramolecular geometries including distortion of the glycosidic valence angle as a function of the glycosidic dihedral angles. The model-compound optimized force field parameters are validated on full disaccharides through comparison of molecular dynamics results to available experimental data. Good agreement is achieved with experiment for a variety of properties including crystal cell parameters and intramolecular geometries, aqueous densities, and aqueous NMR coupling constants associated with the glycosidic linkage. The newly-developed parameters allow for the modeling of linear, branched, and cyclic hexopyranose glycosides both alone and in heterogenous systems including proteins, nucleic acids and/or lipids when combined with existing CHARMM biomolecular force fields.
Project description:We report 100 ns molecular dynamics simulations, at various temperatures, of sucrose in water (with concentrations of sucrose ranging from 0.02 to 4M), and in a 7:3 water-DMSO mixture. Convergence of the resulting conformational ensembles was checked using adaptive-biased simulations along the glycosidic ? and ? torsion angles. NMR relaxation parameters, including longitudinal (R?) and transverse (R?) relaxation rates, nuclear Overhauser enhancements (NOE), and generalized order parameter (S²) were computed from the resulting time-correlation functions. The amplitude and time scales of molecular motions change with temperature and concentration in ways that track closely with experimental results, and are consistent with a model in which sucrose conformational fluctuations are limited (with 80-90% of the conformations having ?-? values within 20° of an average conformation), but with some important differences in conformation between pure water and DMSO-water mixtures.
Project description:Amylose, a component of starch with increasing biotechnological significance, is a linear glucose polysaccharide that self-organizes into single- and double-helical assemblies. Starch granule packing, gelation and inclusion-complex formation result from finely balanced macromolecular kinetics that have eluded precise experimental quantification. Here, graphics processing unit (GPU) accelerated multi-microsecond aqueous simulations are employed to explore conformational kinetics in model single- and double-stranded amylose. The all-atom dynamics concur with prior X-ray and NMR data while surprising and previously overlooked microsecond helix-coil, glycosidic linkage and pyranose ring exchange are hypothesized. In a dodecasaccharide, single-helical collapse was correlated with linkages and rings transitioning from their expected syn and (4)C1 chair conformers. The associated microsecond exchange rates were dependent on proximity to the termini and chain length (comparing hexa- and trisaccharides), while kinetic features of dodecasaccharide linkage and ring flexing are proposed to be a good model for polymers. Similar length double-helices were stable on microsecond timescales but the parallel configuration was sturdier than the antiparallel equivalent. In both, tertiary organization restricted local chain dynamics, implying that simulations of single amylose strands cannot be extrapolated to dimers. Unbiased multi-microsecond simulations of amylose are proposed as a valuable route to probing macromolecular kinetics in water, assessing the impact of chemical modifications on helical stability and accelerating the development of new biotechnologies.
Project description:Cyclic di-nucleotides (CDNs) are second messengers in bacteria and metazoan that are as such controlling important biological processes. Here the conformational space of CDNs was explored systematically by a combination of extensive conformational search and DFT calculations as well as NMR methods. We found that CDNs adopt pre-organized conformations in solution in which the ribose conformations are North type and glycosidic bond conformations are anti type. The overall flexibility of CDNs as well as the backbone torsion angles depend on the cyclization of the phosphodiester bond. Compared to di-nucleotides, CDNs display high rigidity in the macrocyclic moieties. Structural comparison studies demonstrate that the pre-organized conformations of CDNs highly resemble the biologically active conformations. These findings provide information for the design of small molecules to modulate CDNs signalling pathways in bacteria or as vaccine adjuvants. The rigidity of the backbone of CDNs enables the design of high order structures such as molecular cages based on CDNs analogues.