Bisecting βGlcNAc: MA’AT analysis of 13C-labeled oligosaccharides containing βGlcNAc-(1→4)-βMan O-glycosidic linkages

Biologically-relevant mannose- (Man) and N-acetyl-D-glucosamine- (GlcNAc) containing di-, tri-, tetra- and hexasaccharides containing βGlcNAc-(1→4)-βMan O-glycosidic linkages were selectively labeled with ¹³C and used in MA'AT analyses of context effects on linkage conformation and dynamics. Using βGlcNAc-(1→4)-βManOCH₃ as the reference disaccharide, MA'AT analysis provided experiment-based probability distributions for the phi (ϕ) and psi (ψ) torsion angles that comprise its linkage, giving mean values and circular standard deviations, a measure of librational motion about the mean angle, for each angle devoid of context effects. MA'AT analyses of the larger oligosaccharides revealed how the βGlcNAc-(1→4)-βMan linkage behaves conformationally when embedded into larger structures. Context effects on ϕ were generally small (changes in mean values <6°), whereas those on ψ were substantial (changes in mean values up to 25°). Substantial reduction in librational averaging of ψ was observed in the highly-congested hexasaccharide. Similar effects were observed for αMan-(1→3)-βMan linkages, where context effects on ϕ were negligible but those on the mean values of ψ significant (∼14°). The experimental results were compared to those obtained by aqueous molecular dynamics simulation. The results demonstrate the ability of MA'AT analysis to detect and quantify changes in linkage conformational equilibria in solution brought about by structural context, and point to the relative rigidity of ϕ in response to structural crowding compared to ψ, the latter being more responsive to local environment. Embedding a bisecting βGlcNAc residue into biologically-relevant Man-containing oligosaccharides causes substantial change in both linkage conformational preference and librational averaging of linkage torsion angles.