Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.750475
Title: Multidimensional and heteronuclear NMR investigations of oligosaccharides in the free and protein bound states
Author: Milton, Mark John
Awarding Body: University of St Andrews
Current Institution: University of St Andrews
Date of Award: 1997
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Abstract:
The three dimensional structure and dynamics of three biologically important oligosaccharides were probed using a range of NMR techniques. The solution structure of oligosaccharide moiety of was solved using conventional 1H - 1H ROESY data in conjunction with molecular dynamics simulations. It was evident from this study that the dominant conformation in free solution is similar to the structure of oligo-GM1 in complex with the B-subunit of the enteropathogenic cholera toxin. The solution dynamics of an analogue of globoside (Gb4-OEtTMS), a cellular receptor for an E. coli verotoxin, was also elucidated using conventional 1H - 1H ROESY data. However, it was not clear that these data alone could adequately model the structure. Additional distance restraints were derived from 1H - 1H NOE contacts between exchangeable and non-exchangeable protons measured in H2O/acetone-d6 solutions at low temperature (-15 °C). It was concluded on the basis of these studies that Gb4-OEtTMS is an extremely flexible sugar, however, the time- averaged conformation was similar to the proposed conformation of Gb4 in complex with a verotoxin (VT2e). In order to increase the structural information which may be measured from NMR spectroscopy, carbon-13 enriched carbohydrate moieties, commonly found in glycolipids and glycoproteins, were synthesised. Isotopically labelled N-acetyl neuraminic acid, a2,3 sialyl N-acetyl lactosamine (required for the synthesis of sialyl Lewis antigen) and a2,3 sialyl lactose (oligo-GM3), the core ganglioside which is required for the synthesis of more complex gangliosides (e.g. GM1), was accomplished using a mixture of enzymatic and synthetic methods. To test the applicability of heteronuclear NMR spectroscopy in the study of oligosaccharides, the solution structure and dynamics of [U-13C] oligo- GM3 was probed by recording three dimensional 13C-edited ROESY spectra, and long-range carbon-carbon and carbon-proton spin-coupling constants. Inter-residue ROEs could be unambiguously assigned from the 3D experiment, and these assignments contradict previous inter-residue ROE contacts published in the literature (based only on homonuclear data). Two additional inter-residue ROE contacts could be measured across the Gaibeta1-4Glc linkage. The solution conformation of [U-13C] oligo-GM3 in complex with wheat germ agglutinin was solved on the basis of 3D TRNOESY-HSQC data. Theoretical TRNOE intensities back-calculated from full relaxation matrix calculations performed on the X-ray crystal structures of the complex were consistent with experimentally measured values and confirmed the flexible nature of the Galbeta1-4Glc linkage when bound to the protein. Finally, carbon-13 enriched N-acetyl neuraminic acid and galactose were introduced into the glycan chains of an intact glycoprotein FcREA. 13C-1H HSQC data suggest that the two arms of the biantennary glycan experience two distinct magnetic environments.
Supervisor: Homans, Stephen Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.750475  DOI: Not available
Keywords: QP702.S85M5 ; Sugars
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