Solution NMR Structural Studies of Glycans

Abstract

In this account, we present a summary of our progress in the study of glycan solution conformations by NMR spectroscopy. We begin by comparing the structural biology of glycans to that of proteins. We continue by defining the challenges in solution structural studies of glycans as we see them and show what we have done to help address those challenges. This includes Residual Dipolar Coupling studies and their hurdles, accurate coupling constant measurement in the presence of strong 1 H-1 H coupling, direct detection of hydrogen bonds where the NHs, OHs or CHs are the hydrogen bond donors, and on-cell NMR structural studies. We also suggest some future approaches to glycan structure determination.

In-cell NMR: Why and how?

“…Carbohydrates and lipids represent a class of molecules that can clearly benefit from the line-narrowing effects of HR-MAS. Carbohydrates are notoriously difficult to study because of the limited dispersion of their NMR signals [1663][1664][1665][1666]. Moreover, in living cells, polysaccharides are often found in the neighborhood of cell membranes where magnetic susceptibility effects due to heterogeneity are particularly strong, and molecular mobility can be severely hampered.…”

Synthesis of a glycan hairpin

“…Glycan structural studies are even more challenging because the lack of chromophores limits the use of circular dichroism, while difficulties in obtaining single crystals hinder the application of X-ray crystallography to glycans beyond a certain size (that is, tetrasaccharides). NMR may provide some structural information, but suffers from severe overlap between the resonances of different residues and the scarcity of inter-residue and long-range NOEs 26 , 61 , 62 .…”

“…In this Article, we rationally designed a glycan capable of folding autonomously into a secondary structure motif to challenge the common view that sees glycans exclusively as flexible molecules 12 , 26 . Inspired by peptide model systems, we constructed a glycan that adopts a hairpin secondary structure motif in aqueous solution.…”

Growing Glycans in Rosetta: Accurate de novo glycan modeling, density fitting, and rational sequon design

“…While we can generalize that low energy conformations found through the GlycanTreeModeler should be indicative of probable solution conformations, the GlycanTreeModeler was not benchmarked on an experimental ensemble of glycan structures. The few glycan ensembles found through solution NMR [ 58 ] may approximate conformational ensembles in solution and could be the bases for future benchmarking and protocol/scorefunction optimization. However, even with this consideration, many of the benchmark glycans that were modeled accurately to their crystal structures are not hindered by monomer or crystal contacts, but have few interactions to protein residues in their glycan root.…”