The conformational preference of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution has been studied by NMR spectroscopy using one-dimensional (1)H,(1)H T-ROESY experiments and measurement of trans-glycosidic (3)J(C,H) coupling constants. Molecular dynamics (MD) simulations with a CHARMM22 type of force field modified for carbohydrates were performed with water as the explicit solvent. The homonuclear cross-relaxation rates, interpreted as effective proton-proton distances, were compared to those obtained from simulation. Via a Karplus torsional relationship, (3)J(C,H) values were calculated from simulation and compared to experimental data. Good agreement was observed between experimental data and the MD simulation, except for one inter-residue T-ROE between protons in the terminal sugar residues. The results show that the trisaccharide exhibits substantial conformational flexibility, in particular along the psi glycosidic torsion angles. Notably, for these torsions, a high degree of correlation (77%) was observed in the MD simulation revealing either psi(2)(+) psi(3)(+) or psi(2)(-)psi(3)(-) states. The simulations also showed that non-exoanomeric conformations were present at the phi torsion angles, but to a limited extent, with the phi(3) state populated to a larger extent than the phi(2) state. Further NMR analysis of the trisaccharide by translational diffusion measurements and (13)C T(1) relaxation experiments quantified global reorientation using an anisotropic model together with interpretation of the internal dynamics via the "model-free" approach. Fitting of the dynamically averaged states to experimental data showed that the psi(2)(+)psi(3)(+) state is present to approximately 49%, psi(2)(-) psi(3)(-) to approximately 39%, and phi(3) (non-exo) to approximately 12%. Finally, using a dynamic and population-averaged model, (1)H,(1)H T-ROE buildup curves were calculated using a full relaxation matrix approach and were found to be in excellent agreement with experimental data, in particular for the above inter-residue proton-proton interaction between the terminal residues.
The beta-D-GlcpNAc-(1-->6)-alpha-D-Manp disaccharide is a constituent of highly branched cell-surface glycoconjugates that are malignancy markers. The conformational preference of the disaccharide beta-D-GlcpNAc-(1-->6)-alpha-D-Manp-OMe in solution has been studied by molecular modeling and NMR spectroscopy including 1D (1)H,(1)H T-ROESY experiments and analysis of (3)J(H,H) of the hydroxymethyl group being part of the glycosidic linkage of the disaccharide, which revealed the relative populations of the omega torsion angle as gt = 0.60, gg = 0.35, and tg = 0.05. Good agreement was obtained between the effective proton-proton distances from the experiment and those obtained by molecular modeling when the flexibility at the omega torsion angle was taken into account. Molecular modeling of the disaccharide in the binding sites of the lectin wheat germ agglutinin indicates that several conformations could be adopted in the bound state. (1)H NMR and transfer NOESY experiments confirmed that binding took place, and trans-glycosidic proton-proton interactions indicated that a conformational preference was present in the bound state, as observed by the relative change of the NOEs from H1' to H6(pro-R) and H6(pro-S). STD NMR experiments showed that binding occurred in the region of the N-acetyl group of the terminal sugar residue. In addition, the O-methyl group received saturation transfer because of the proximity to the protein. (1)H,(1)H NOEs indicated that the two methyl groups were close in space, as observed in only one of the predicted bound conformations. Experimental and theoretical data therefore agree that one conformation with a gt conformation of the hydroxymethyl group and a negative sign for the psi torsion angle is indeed selected by the lectin upon binding.
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