The Incorporation of Labile Protons into Multidimensional NMR Analyses: Glycan Structures Revisited

Abstract

Glycan structures are often stabilized by a repertoire of hydrogen-bonded donor/acceptor groups, revealing longerlived structures that could represent biologically relevant conformations. NMR provides unique data on these hydrogen-bonded networks from multidimensional experiments detecting cross-peaks resulting from through-bond (TOCSY) or through-space (NOESY) interactions. However, fast OH/H 2 O exchange, and the spectral proximity among these NMR resonances, hamper the use of glycans' labile protons in such analyses; consequently, studies are often restricted to aprotic solvents or supercooled aqueous solutions. These nonphysiological conditions may lead to unrepresentative structures or to probing a small subset of accessible conformations that may miss "active" glycan conformations. Looped, projected spectroscopy (L-PROSY) has been recently shown to substantially enhance protein NOESY and TOCSY cross-peaks, for 1 Hs that undergo fast exchange with water. This study shows that even larger enhancements can be obtained for rapidly exchanging OHs in saccharides, leading to the retrieval of previously undetectable 2D TOCSY/NOESY cross-peaks with nonlabile protons. After demonstrating ≥300% signal enhancements on model monosaccharides, these experiments were applied at 1 GHz to elucidate the structural network adopted by a sialic acid homotetramer, used as a model for α,2−8 linked polysaccharides. High-field L-PROSY NMR enabled these studies at higher temperatures and provided insight previously unavailable from lower-field NMR investigations on supercooled samples, involving mostly nonlabile nuclei. Using L-PROSY's NOEs and other restraints, a revised structural model for the homotetramer was obtained combining rigid motifs and flexible segments, that is well represented by conformations derived from 40 μs molecular dynamics simulations.

Molecular Recognition Insights of Sialic Acid Glycans by Distinct Receptors Unveiled by NMR and Molecular Modeling

“…Therefore, it was hypothesized that PolySia structures with a certain degree of polymerization adopt a specific conformation in solution that would promote the binding of antibodies ( Azurmendi et al, 2017 ). In this regard, several conformational studies were carried out on PolySia with different degrees of polymerization ( Henderson et al, 2003 ; Battistel et al, 2012 ; Hanashima et al, 2013 ; Turupcu et al, 2020 ; Novakovic et al, 2021 ). However, no consensus is found in the literature regarding the PolySia conformation, which can alternate between random coil and distinct types of helical conformations.…”

“… 2-D structure representation of (Sia) 4 with the most important structural features deduced by Battistel et al, 2012 (A) and Novakovic et al, 2021 (B). (A) .…”

“…In blue dashed lines are the H-bonds IIO1-IOH2, consistent with a rigid conformation in the reducing end of (Sia) 4 , and IVOH8-IVO1 the principal structural motif for the most flexible part of the nonreducing end. Figures adapted from Battistel et al (2012 ) and Novakovic et al (2021 ).…”

“…Recently, high-field Looped-PROjected spectroscopy (L-PROSY) NMR was employed to revisit the (Sia) 4 conformation closer to physiological conditions in a solution at 278 K ( Novakovic et al, 2021 ). This experiment enhances the NOESY and TOCSY cross-peaks for 1 Hs that undergo fast exchange with water, and it is ideal to extract structural information from hydroxyl groups in sugars.…”

“…This structural motif was previously observed in shorter (Sia) 2 homologue, and it was suggested to be a common feature of PolySia structures ( Azurmendi et al, 2017 ). For the nonreducing end (Sia III-IV residues), the authors reported high potential flexibility together with the typical structural motif of the glycerol side chain of the terminal end Sia IV, characterized by the stable intra-residue H-bond between OH 8 and O1 ( Novakovic et al, 2021 ). Notably, a direct through-hydrogen-bond correlation was detected via long-range HSQMBC between OH 8 and C1, compatible with a stable H-bond between OH8 and O1 of Sia IV ( Figure 2B ).…”