NMR structure analysis of uniformly 13C-labeled carbohydrates
In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of 13C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly 13C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-13C)-sucrose, 342 Da] and one compound of medium molecular weight (13C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The 13C resonances are traced using 13C–13C correlations from homonuclear experiments, such as (H)CC–CT–COSY, (H)CC–NOESY, CC–CT–TOCSY and/or virtually decoupled (H)CC–TOCSY. Based on the assignment of the 13C resonances, the 1H chemical shifts are derived in a straightforward manner using one-bond 1H–13C correlations from heteronuclear experiments (HC–CT–HSQC). In order to avoid the 1 J CC splitting of the 13C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either 13C or 1H detected experiments, namely CC–CT–COSY, band-selective (H)CC–TOCSY, HC–CT–HSQC–NOESY or long-range HC–CT–HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the 1H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the 13C-enriched polysaccharide were assigned by using HC–H2BC spectra. The assignment of the N-acetyl groups with 15N at natural abundance was completed by using HN–SOFAST–HMQC, HNCA, HNCO and 13C-detected (H)CACO spectra.
KeywordsCarbohydrates 13C-uniform labeling NMR Structure determination
This work was supported by grants from the Swedish Research Council and the Knut and Alice Wallenberg Foundation. The research that has led to these results has received funding from the European Commission’s Seventh Framework Programme FP7/2007–2013 under grant agreement no. 215536.
- Bugarel M, Martin A, Fach P, Beutin L (2011) Virulence gene profiling of enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains: a basis for molecular risk assessment of typical and atypical EPEC strains. BMC Microbiol 11:142. doi: 10.1186/1471-2180-11-142 CrossRefGoogle Scholar
- Fairweather JK, Him JLK, Heux L, Driguez H, Bulone V (2004) Structural characterization by 13C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using 13C-enriched glycosyl donors: application to a UDP-glucose:(1 → 3)-β-d-glucan synthase from blackberry (Rubus fructicosus). Glycobiology 14:775–781. doi: 10.1093/glycob/cwh097 CrossRefGoogle Scholar
- Kjellberg A, Nishida T, Weintraub A, Widmalm G (1998) NMR spectroscopy of 13C-enriched polysaccharides: application of 13C–13C TOCSY to sugars of different configuration. Magn Reson Chem 36:128–131. doi: 10.1002/(SICI)1097-458X(199802)36:2<128:AID-OMR226>3.0.CO;2-L CrossRefGoogle Scholar
- Linnerborg M, Weintraub A, Widmalm G (1999) Structural studies utilizing 13C-enrichment of the O-antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross-reacting with Shigella dysenteriae type 4. Eur J Biochem 266:246–251. doi: 10.1046/j.1432-1327.1999.00851.x CrossRefGoogle Scholar
- Martin-Pastor M, Bush CA (2000) Comparison of the conformation and dynamics of a polysaccharide and of its isolated heptasaccharide repeating unit on the basis of nuclear Overhauser effect, long-range C–C and C–H coupling constants, and NMR relaxation data. Biopolymers 54:235–248. doi: 10.1002/1097-0282(20001005)54:4<235:AID-BIP10>3.0.CO;2-V CrossRefGoogle Scholar
- Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (2009) Essentials of glycobiology, vol 2. Cold Spring Harbor, New YorkGoogle Scholar
- Villeneuve S, Souchon H, Riottot M–M, Mazié J-C, Lei P-s, Glaudemans CPJ, Kováč P, Fournier J-M, Alzari PM (2000) Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity. Proc Natl Acad Sci U S A 97:8433–8438. doi: 10.1073/pnas.060022997 CrossRefADSGoogle Scholar