Transient hydrogen bonding in uniformly ¹³C,¹⁵N‐Labeled Carbohydrates in Water

Abstract

We report NMR studies of transient hydrogen bonding in a polysaccharide (PS) dissolved in water without cosolvent at ambient temperature. The PS portion of the Escherichia coli O142 lipopolysaccharide is comprised of repeating pentasaccharide units of GalNAc (N-acetyl galactosamine), GlcNAc (N-acetyl glucosamine), and rhamnose in a 3:1:1 ratio, respectively. A 105-ns molecular dynamics (MD) simulation on one pentasaccharide repeat unit predicts transient inter-residue hydrogen bonds from the GalNAc NH groups in the PS. To investigate these predictions experimentally, the PS was uniformly ¹³C,¹⁵N enriched and the NH, carbonyl, C2, C4, and methyl resonances of the GalNAc and GlcNAc residues assigned using through-bond triple-resonance NMR experiments. Temperature dependence of amide NH chemical shifts and one-bond NH J couplings support that NH groups on two of the GalNAc residues are donors in transient hydrogen bonds. The remaining GalNAc and GlcNAc NHs do not appear to be donors from either temperature-dependent chemical shifts or one-bond NH J couplings. These results substantiate the presence of weak or partial hydrogen bonds in carbohydrates, and that MD simulations of repeating units in PSs provide insight into overall PS structure and dynamics.

Dynamics of exocyclic groups in the Escherichia coli O91 O-antigen polysaccharide in solution studied by carbon-13 NMR relaxation

“…The structure and dynamics of polysaccharides are being studied by many different biophysical techniques (Carriere et al 1993;Tang et al 2004;Chytil et al 2010;Jasnin et al 2010) and NMR spectroscopy is an important one to this end (Hills et al 1991;Hricovini et al 1997;Vlachou et al 2001;Henderson et al 2003). Like for NMR investigations of proteins and nucleic acids the use of 13 C-and/or 15 N-isotope enrichment is a powerful approach to obtain high quality data for polysaccharides in solution (Gitti et al 1994;Kjellberg et al 1999;Norris et al 2012). …”

Inter‐residual Hydrogen Bonding in Carbohydrates Unraveled by NMR Spectroscopy and Molecular Dynamics Simulations

“…Temperature variation combined with analysis of the NMR spectroscopic variables such as spin–spin coupling constants or chemical shifts may unravel atomic interactions within molecules. For the O‐antigen from E. coli O142, we previously analyzed Δ 1 J NH /Δ T values for the d ‐GlcNAc and d ‐GalNAc residues in the repeating unit of the polysaccharide and found that the effect was small and negative for two of them, but was significant and negative for the other two . The temperature dependence of the one‐bond NH coupling in 1 was measured in the temperature range 5—25 °C, but Δ 1 J NH /Δ T ≈0 Hz K −1 and no further conclusion could be drawn for this analysis.…”

NMR structure analysis of uniformly 13C-labeled carbohydrates

“…In the case of E. coli, 174 serogroups are currently described and some of them are considered pathogenic, such is the case of the aforementioned E. coli O142 and O91 serogroups which have been classified as enteropathogenic E. coli (EPEC) (Bugarel et al 2011) and Shiga toxing-producing E. coli (STEC) (Son et al 2014), respectively. Analogously to what is found in proteins and nucleic acids, the NH groups of amino sugars can also act as hydrogen donors and influence the three-dimensional structure of carbohydrates through inter-residue hydrogen bonding interactions (Norris et al 2012). Furthermore, the many hydroxyl groups available in these molecules can act either as hydrogen donors or acceptors but, since the H2O molecules can also compete for the same hydrogen bond, the use of aprotic co-solvents and/or low temperatures may be required to detect such kind of interactions (Battistel et al 2013;Norris et al 2012).…”

“…Analogously to what is found in proteins and nucleic acids, the NH groups of amino sugars can also act as hydrogen donors and influence the three-dimensional structure of carbohydrates through inter-residue hydrogen bonding interactions (Norris et al 2012). Furthermore, the many hydroxyl groups available in these molecules can act either as hydrogen donors or acceptors but, since the H2O molecules can also compete for the same hydrogen bond, the use of aprotic co-solvents and/or low temperatures may be required to detect such kind of interactions (Battistel et al 2013;Norris et al 2012). Whether hydrogen bonds involving hydroxyl groups may or may not influence the threedimensional fold of carbohydrates under physiological conditions is a question that still remains unanswered; they have, however, proven to play a critical role in carbohydrate-protein interactions such as in the recognition by antibodies (Villeneuve et al 2000).…”

“…The use of 13 C-detected experiments is well established in the NMR spectroscopy of 13 C-enriched proteins (Bermel et al 2008;Bermel et al 2006) and nucleic acids (Farès et al 2007;Fiala and Sklenár 2007;Richter et al 2010), but only limited use has been made of 13 C-enriched carbohydrates (Battistel et al 2012;Harris et al 1997; Kiddle and Homans 1998;Kjellberg et al 1998;Martin-Pastor et al 2003;Martin-Pastor and Bush 2000;Norris et al 2012;Wang et al 2008;Xu and Bush 1998;Yu et al 1993). Glycans do not necessarily assume a particular fold free in solution, instead, they may appear in extended, flexible conformations with mere transient structural elements (Martin-Pastor and Bush 2000;Sarkar et al 2013).…”