A dynamic perspective on the molecular recognition of chitooligosaccharide ligands by hevein domains

The complexes between hevein and different chitin oligomers, from the di- to the penta-saccharide, are studied through all atom molecular-dynamics simulations. The results for the smaller oligosaccharide complexes show that the carbohydrate is able to move on the surface of the relatively flat binding-pocket of hevein, therefore occupying different binding subpockets. The pentasaccharide spans all possible intermolecular interactions with the receptor in a simultaneous manner. Statistical analysis methods were also applied in order to define the principal overall motions in the complexes. The oligosaccharide binding can be considered to be defined by a subtle balance between enthalpic and entropic effects, providing the possibility of the existence of multiple binding conformations. This structural and dynamical view parallels the results based on NOE NMR data for the three disaccharide, trisaccharide, and pentasaccharide complexes.     

NMR investigations of protein-carbohydrate interactions: studies on the relevance of Trp/Tyr variations in lectin binding sites as deduced from titration microcalorimetry and NMR studies on hevein domains. Determination of the NMR structure of the complex between pseudohevein and N,N',N"-triacetylchitotriose

Model studies on lectins and their interactions with carbohydrate ligands in solution are essential to gain insights into the driving forces for complex formation and to optimize programs for computer simulations. The specific interaction of pseudohevein with N,N', N"-triacetylchitotriose has been analyzed by (1)H-NMR spectroscopy. Because of its small size, with a chain length of 45 amino acids, this lectin is a prime target to solution-structure determination by NOESY NMR experiments in water. The NMR-analysis was extended to assessment of the topology of the complex between pseudohevein and N, N',N"-triacetylchitotriose. NOESY experiments in water solution provided 342 protein proton-proton distance constraints. Binding of the ligand did not affect the pattern of the protein nuclear Overhauser effect signal noticeably, what would otherwise be indicative of a ligand-induced conformational change. The average backbone (residues 3-41) RMSD of the 20 refined structures was 1.14 A, whereas the heavy atom RMSD was 2.18 A. Two different orientations of the trisaccharide within the pseudohevein binding site are suggested, furnishing an explanation in structural terms for the lectin's capacity to target chitin. In both cases, hydrogen bonds and van der Waals contacts confer stability to the complexes. This conclusion is corroborated by the thermodynamic parameters of binding determined by NMR and isothermal titration calorimetry. The association process was enthalpically driven. In relation to hevein, the Trp/Tyr-substitution in the binding pocket has only a small effect on the free energy of binding in contrast to engineered galectin-1 and a mammalian C-type lectin. A comparison of the three-dimensional structure of pseudohevein in solution to those reported for wheat germ agglutinin (WGA) in the solid state and for hevein and WGA-B in solution has been performed, providing a data source about structural variability of the hevein domains. The experimentally derived structures and the values of the solvent accessibilities for several key residues have also been compared with conformations obtained by molecular dynamics simulations, pointing to the necessity to further refine the programs to enhance their predictive reliability and, thus, underscoring the importance of this kind of combined analysis in model systems.     

NMR investigations of protein-carbohydrate interactions binding studies and refined three-dimensional solution structure of the complex between the B domain of wheat germ agglutinin and N,N', N"-triacetylchitotriose.

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA-B) with N,N',N"-triacetylchitotriose has been analyzed by 1H-NMR spectroscopy. The association constants for the binding of WGA-B to this trisaccharide have been determined from both 1H-NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative DeltaH which is partially compensated by negative DeltaS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton-proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 A, while the heavy atom rmsd was 2.10 A. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA-B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three-dimensional structure of WGA-B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.     

Fluorescence polarization studies of saccharide binding to Wheat germ agglutinin and lysozyme

N-acetyl-D-glucosamine causes only slight increases in the polarization of Wheat germ agglutinin fluorescence at saturating levels whereas the disaccharide and trisaccharide produce increases in the polarization value from 0.116 to 0.151 and 0.154 respectively. These increases suggest that rotational motions of the tryptophan residue at the binding sites are being restricted by an interaction between these tryptophans and the bound sugars. A model of the nature and location of these interactions is discussed.Comparable results are obtained with lysozyme, which shows a larger effect upon binding of N-acetyl-D-glucosamine, but a maximal increase in polarization upon binding the corresponding disaccharide or trisaccharide.     

Comprehensive analysis of motions in molecular dynamics trajectories of the actin capping protein and its inhibitor complexes

The actin capping protein (CP) binds to actin filaments to block further elongation. The capping activity is inhibited by proteins V‐1 and CARMIL interacting with CP via steric and allosteric mechanisms, respectively. The crystal structures of free CP, CP/V‐1, and CP/CARMIL complexes suggest that the binding of CARMIL alters the flexibility of CP rather than the overall structure of CP, and this is an allosteric inhibition mechanism. Here, we performed molecular dynamics (MD) simulations of CP in the free form, and in complex with CARMIL or V‐1. The resulting trajectories were analyzed exhaustively using Motion Tree, which identifies various rigid‐body motions ranging from small local motions to large domain motions. After enumerating all the motions, CP flexibilities with different ligands were characterized by a list of frequencies for 20 dominant rigid‐body motions, some of which were not identified in previous studies. The comparative analysis highlights the influence of the binding of the CARMIL peptide to CP flexibility. In free CP and the CP/V‐1 complex, domain motions around a large crevice between the N‐stalk and the CP‐S domain occur frequently. The CARMIL peptide binds the crevice and suppresses the motions effectively. In addition, the binding of the CARMIL peptide enhances and alters local motions around the pocket that participates in V‐1 binding. These newly identified motions are likely to suppress the binding of V‐1 to CP. The observed changes in CP motion provide insights that describe the mechanism of allosteric regulation by CARMIL through modulating CP flexibility. Proteins 2016; 84:948–956. © 2016 Wiley Periodicals, Inc.     

Molecular dynamics simulation of Lewis blood groups and related oligosaccharides.

Molecular dynamics simulations without explicit inclusion of solvent molecules have been performed to study the motions of Lewisa and Lewisb blood group oligosaccharides, and two blood group A tetrasaccharides having type I and type II core chains. The blood group H trisaccharide has also been studied and compared with the blood group A type II core chain. The potential energy surface developed by Rasmussen and co-workers was used with the molecular mechanics code CHARMM. The lowest energy minima of the component disaccharide fragments were obtained from conformational energy mapping. The lowest energy minima of these disaccharide fragments were used to build the tri- and tetrasaccharides that were further minimized before the actual heating/equilibration and dynamics simulations. The trajectories of the disaccharide fragments, e.g., Fuc alpha- (1----4)GlcNAc, Gal beta-(1----4)GlcNAc, etc., show transitions among various minima. However, the oligosaccharides were found to be dynamically stable and no transitions to other minimum energy conformations were observed in the time series of the glycosidic dihedral angles even during trajectories as long as 300 ps. The stable conformations of the glycosidic linkages in the oligosaccharides are not necessarily the same as the minimum energy conformation of the corresponding isolated disaccharides. The average fluctuations of the glycosidic angles in the oligosaccharides were well within the range of +/- 15 degrees. The results of these trajectory calculations were consistent with the relatively rigid single-conformation models derived for these oligosaccharides from 1H-nmr data.     

Regional Central Nervous System Oligosaccharide Storage in Caprine β-Mannosidosis

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.     

Molecular dynamics simulations of galectin-1-oligosaccharide complexes reveal the molecular basis for ligand diversity

Galectin-1 is a member of a protein family historically characterized by its ability to bind carbohydrates containing a terminal galactosyl residue. Galectin-1 is found in a variety of mammalian tissues as a homodimer of 14.5-kDa subunits. A number of developmental and regulatory processes have been attributed to the ability of galectin-1 to bind a variety of oligosaccharides containing the Gal-beta-(1,4)-GlcNAc (LacNAc(II)) sequence. To probe the origin of this permissive binding, solvated molecular dynamics (MD) simulations of several representative galectin-1-ligand complexes have been performed. Simulations of structurally defined complexes have validated the computational approach and expanded upon data obtained from X-ray crystallography and surface plasmon resonance measurements. The MD results indicate that a set of anchoring interactions between the galectin-1 carbohydrate recognition domain (CRD) and the LacNAc core are maintained for a diverse set of ligands and that substituents at the nonreducing terminus of the oligosaccharide extend into the remainder of a characteristic surface groove. The anionic nature of ligands exhibiting relatively high affinities for galectin-1 implicates electrostatic interactions in ligand selectivity, which is confirmed by a generalized Born analysis of the complexes. The results suggest that the search for a single endogenous ligand or function for this lectin may be inappropriate and instead support a more general role for galectin-1, in which the lectin is able to crosslink heterogeneous oligosaccharides displayed on a variety of cell surfaces. Such binding promiscuity provides an explanation for the variety of adhesion phenomena mediated by galectin-1.     

Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment

The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.     

Conformation of a trimannoside bound to mannose-binding protein by nuclear magnetic resonance and molecular dynamics simulations

A model of the carbohydrate recognition domain of the serum form of mannose-binding protein (MBP) from rat complexed with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside is presented. Allowed conformations for the bound sugar were derived from simulated annealing protocols incorporating distance restraints computed from transferred NOESY spectra. The resulting sugar conformations were then modeled into the MBP binding site, and these models of the complex were refined using molecular dynamics (MD) simulations in the presence of solvent water. These studies indicate that only one of the two major conformations of the alpha(1-->6) linkage found in solution is significantly populated in the bound state (omega = 60 degrees ), whereas the alpha(1-->3) linkage samples at least two states, similar to its behavior in free solution. The bound conformation allows direct hydrogen bonds to form between the sugar and K182 of MBP, in addition to other water-mediated hydrogen bonds. Estimates of binding constants of candidate complexes based on changes in solvent-accessible surface areas upon binding support the NMR and MD results. These estimates further suggest that the enthalpic gains of the additional sugar-MBP interactions in a trisaccharide as opposed to a monosaccharide are offset by entropic penalties, offering an explanation for previous binding data.     

Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres

The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions. The Pot1 amino-terminal DNA binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity. We have now examined DNA binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein. Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection. Specifically, full-length Pot1.DNA complexes are more stable, and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats. We provide evidence that Pot1 contains a second DNA binding motif that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate, whereby the amino-terminal oligonucleotide/oligosaccharide binding fold determines the registry of binding, and the internal DNA binding motif stabilizes the complex and expands the protected region toward the 3' -end. Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3'-end and protects against exonucleolytic degradation.     

Investigations of peptide hydration using NMR and molecular dynamics simulations: A study of effects of water on the conformation and dynamics of antamanide

Summary The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 s at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight bound water molecules. Within the simulation time of 600 ps, no water molecule leaves the complex. Additionally, the simulations show a reduced flexibility for the complex in comparison with uncomplexed antamanide. Thus, there is a qualitative agreement between the experimental NMR results and the computer simulations.     

Structural basis of the preferential binding for globo-series glycosphingolipids displayed by Pseudomonas aeruginosa lectin I

The opportunistic pathogen Pseudomonas aeruginosa contains several carbohydrate-binding proteins, among which is the P. aeruginosa lectin I (PA-IL), which displays affinity for alpha-galactosylated glycans. Glycan arrays were screened and demonstrated stronger binding of PA-IL toward alphaGal1-4betaGal-terminating structures and weaker binding to alphaGal1-3betaGal ones in order to determine which human glycoconjugates could play a role in the carbohydrate-mediated adhesion of the bacteria. This was confirmed in vivo by testing the binding of the lectin to Burkitt lymphoma cells that present large amounts of globotriaosylceramide antigen Gb3/CD77/P(k). Trisaccharide moieties of Gb3 (alphaGal1-4betaGal1-4Glc) and isoglobotriaosylceramide (alphaGal1-3betaGal1-4Glc) were tested by titration microcalorimetry, and both displayed similar affinity to PA-IL in solution. The crystal structure of PA-IL complexed to alphaGal1-3betaGal1-4Glc trisaccharide has been solved at 1.9-A resolution and revealed how the second galactose residue makes specific contacts with the protein surface. Molecular modeling studies were performed in order to compare the binding mode of PA-IL toward alphaGal1-3Gal with that toward alphaGal1-4Gal. Docking studies demonstrated that alphaGal1-4Gal creates another network of contacts for achieving a very similar affinity, and 10-ns molecular dynamics in explicit water allowed for analyzing the flexibility of each disaccharide ligand in the protein binding site. The higher affinity observed for binding to Gb3 epitope, both in vivo and on glycan array, is likely related to the presentation effect of the oligosaccharide on a surface, since only the Gb3 glycosphingolipid geometry is fully compatible with parallel insertion of neighboring trisaccharide heads in two binding sites of the same tetramer of PA-IL.     

On the Information Content of Protein Sequences

Abstract

TATA-box binding protein (TBP) in a monomelic form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in ‘flipflop’ contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.     

Single-molecule fluorescence measurements of ribosomal translocation dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.     

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation.

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.     

Collective dynamics of EcoRI-DNA complex by elastic network model and molecular dynamics simulations

Anisotropic network model (ANM) is used to analyze the collective motions of restriction enzyme EcoRI in free form and in complex with DNA. For comparison, three independent molecular dynamics (MD) simulations, each of 1.5 ns duration, are also performed for the EcoRI-DNA complex in explicit water. Although high mobility (equilibrium fluctuations) of inner and outer loops that surround the DNA is consistent in both methods and experiments, MD runs sample different conformational subspaces from which reliable collective dynamics cannot be extracted. However, ANM employed on different conformations from MD simulations indicates very similar collective motions. The stems of the inner loops are quite immobile even in the free enzyme and form a large, almost fixed, pocket for DNA binding. As a result, the residues that make specific and non-specific interactions with the DNA exhibit very low fluctuations in the free enzyme. The vibrational entropy difference between the EcoRI complex and free protein + unkinked DNA is positive (favorable), which may partially counteract the unfavorable enthalpy difference of DNA kink formation. Dynamic domains in EcoRI complex and cross-correlations between residue fluctuations indicate possible means of communication between the distal active sites.     

NMR investigations of protein-carbohydrate interactions: refined three- dimensional structure of the complex between hevein and methyl beta- chitobioside

The specific interaction of hevein with GlcNAc-containing oligosaccharides has been analyzed by1H-NMR spectroscopy. The association constants for the binding of hevein to a variety of ligands have been estimated from1H-NMR titration experiments. The association constants increase in the order GlcNAc-alpha(1-->6)-Man < GlcNAc < benzyl-beta-GlcNAc < p-nitrophenyl-beta-GlcNAc < chitobiose < p- nitrophenyl-beta-chitobioside < methyl-beta-chitobioside < chitotriose. Entropy and enthalpy of binding for different complexes have been obtained from van't Hoff analysis. The driving force for the binding process is provided by a negative DeltaH0which is partially compensated by negative DeltaS0. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 475 accurate protein proton-proton distance constraints after employing the MARDIGRAS program. In addition, 15 unambiguous protein/carbohydrate NOEs were detected. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the highly refined solution conformation of this protein- carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein nOe's were observed, indicating that carbohydrate-induced conformational changes are small. The average backbone rmsd of the 20 refined structures was 0.055 nm, while the heavy atom rmsd was 0.116 nm. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to the complex. A comparison of the three-dimensional structure of hevein in solution to those reported for wheat germ agglutinin (WGA) and hevein itself in the solid state has also been performed. The polypeptide conformation has also been compared to the NMR-derived structure of a smaller antifungical peptide, Ac-AMP2.      

Molecular dynamics simulations of alpha2 --> 8-linked disialoside: conformational analysis and implications for binding to proteins.

Computational methods have played a key role in elucidating the various three-dimensional structures of oligosaccharides. Such structural information, together with other experimental data, leads to a better understanding of the role of oligosaccharide in various biological processes. The disialoside Neu5Ac-alpha2-->8-Neu5Ac appears as the terminal glycan in glycoproteins and glycolipids, and is known to play an important role in various events of cellular communication. Neurotoxins such as botulinum and tetanus require Neu5Ac-alpha2 --> 8-Neu5Ac for infecting the host. Glycoconjugates containing this disialoside and the enzymes catalyzing their biosynthesis are also regulated during cell growth, development, and differentiation. Unlike other biologically relevant disaccharides that have only two linkage bonds, the alpha2-->8-linked disialoside has four: C2-O, O-C8', C8'-C7', and C7'-C6'. The present report describes the results from nine 1 ns MD simulations of alpha2-->8-linked disialoside (Neu5Ac-alpha2-->8-Neu5Ac); simulations were run using GROMOS96 by explicitly considering the solvent molecules. Conformations around the O-C8' bond are restricted to the +sc/+ap regions due to stereochemical reasons. In contrast, conformations around the C2-O and C8'-C7' bonds were found to be largely unrestricted and all the three staggered regions are accessible. The conformations around the C7'-C6' bond were found to be in either the -sc or the anti region. These results are in excellent agreement with the available NMR and potential energy calculation studies. Overall, the disaccharide is flexible and adopts mainly two ensembles of conformations differing in the conformation around the C7'-C6' bond. The flexibility associated with this disaccharide allows for better optimization of intermolecular contacts while binding to proteins and this may partially compensate for the loss of conformational entropy that may be incurred due to disaccharide's flexibility.