Exploration of Conformational Spaces of High‐Mannose‐Type Oligosaccharides by an NMR‐Validated Simulation

Abstract

Exploration of the conformational spaces of flexible biomacromolecules is essential for quantitatively understanding the energetics of their molecular recognition processes. We employed stable isotope- and lanthanide-assisted NMR approaches in conjunction with replica-exchange molecular dynamics (REMD) simulations to obtain atomic descriptions of the conformational dynamics of high-mannose-type oligosaccharides, which harbor intracellular glycoprotein-fate determinants in their triantennary structures. The experimentally validated REMD simulation provided quantitative views of the dynamic conformational ensembles of the complicated, branched oligosaccharides, and indicated significant expansion of the conformational space upon removal of a terminal mannose residue during the functional glycan-processing pathway.

Paramagnetic NMR probes for characterization of the dynamic conformations and interactions of oligosaccharides

“…This demonstrates that paramagnetism-assisted NMR could be applicable to the determination of the 3D conformations of oligosaccharides in solution, if the molecule could be sufficiently rigid. Furthermore, the introduction of lanthanide tags causes distinct PCSs even for identical non-reducing terminal groups in multiantenary oligosaccharides, resolving their severely overlapping peaks and facilitating spectral analysis [60,61].…”

“…PCS-based conformational analysis has also been applied to complex-type and high-mannose-type oligosaccharides [60,61]. A triantennary oligosaccharide containing nine mannose residues, M9, as well as its derivative M8B was overexpressed in genetically engineered yeast cells as fully 13 C-labeled forms [62,63] and attached to an EDTA-based lanthanide-chelating tag (Fig.…”

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch‐Selective Interaction of a Tetra‐Antennary N‐Glycan with Lectins

“…[6] This methodology has first been applied to the study of small oligosaccharides (di-, tri-, and tetrasaccharides), [7][8][9][10][11] then to N-glycans.T he conformational properties of complex-type bi-antennary and high-mannosetype N-glycans were elucidated and could be resolved in each case down to the level of individual branches. [4,5] Proceeding from this experimental basis,weherein extend this concept to the level of high-degree branching,and show that it is possible to experimentally characterize the conformational behavior and recognition properties of ag alactosylated complex-type tetra-antennary N-glycan, which is not possible by standard NMR methods owing to chemical shift degeneracyo ft he glycan. We have then tested the hypothesis of branch selectivity for two N-acetyllactosamine-binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA1 20).…”

“…[4,5] Proceeding from this experimental basis,weherein extend this concept to the level of high-degree branching,and show that it is possible to experimentally characterize the conformational behavior and recognition properties of ag alactosylated complex-type…”

“…As ap romising approach, carbohydrates conjugated to lanthanide-binding tags have shown high potential toward this aim. [4,5] In its vicinity,acomplexed paramagnetic ion induces significant chemical shift changes of the NMR signals of the glycan as ar esult of dipolar interactions involving the unpaired electron of the metal. These pseudocontact shifts (PCS) depend on the distance between each proton and the metal (proportional to 1/r 3 ).…”