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NMR structure analysis of uniformly 13C-labeled carbohydrates
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.ORCID iD: 0000-0001-8303-4481
2014 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 59, no 2, p. 95-110Article in journal (Refereed) Published
Abstract [en]

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of C-13-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly C-13-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-C-13)-sucrose, 342 Da] and one compound of medium molecular weight (C-13-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, similar to 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 C-13 resonances are traced using C-13-C-13 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 C-13 resonances, the H-1 chemical shifts are derived in a straightforward manner using one-bond H-1-C-13 correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J (CC) splitting of the C-13 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 C-13 or H-1 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 T-2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the H-1-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the C-13-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with N-15 at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and C-13-detected (H)CACO spectra.

Place, publisher, year, edition, pages
2014. Vol. 59, no 2, p. 95-110
Keyword [en]
Carbohydrates, C-13-uniform labeling, NMR, Structure determination
National Category
Biochemistry and Molecular Biology Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-105413DOI: 10.1007/s10858-014-9830-6ISI: 000336399400005OAI: oai:DiVA.org:su-105413DiVA, id: diva2:729023
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:3;

Available from: 2014-06-25 Created: 2014-06-24 Last updated: 2018-03-14Bibliographically approved
In thesis
1. NMR spectroscopy in structural and conformational analysis of bacterial polysaccharides
Open this publication in new window or tab >>NMR spectroscopy in structural and conformational analysis of bacterial polysaccharides
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Carbohydrates constitute one of the three major classes of biomolecules found in all living cells and, unlike nucleic acids and proteins, their polymeric structures are not based on a template. The structural diversity of these molecules confers them an enormous capacity to encode information in biological systems, acting as efficient mediators in the interaction of the cell with the environment. In order to understand the roles of glycans in biological processes it is of key importance to have a detailed understanding of their structures and conformational preferences, and NMR spectroscopy is one of most powerful techniques for the study of these molecules in solution.

This thesis is focused on the structural and conformational analysis of lipopolysaccharides from Gram-negative bacteria. In the first two projects (Chapter 2 and 3) the structural analyses of the biological repeating units of the O-antigen polysaccharides from E. coli O174ab and O115 are described; in both cases a combination of NMR spectroscopy and gas chromatography techniques were used. Special emphasis was made in the characterization of the O-acetylation patterns observed in the native O-antigen polysaccharide from E. coli O115. Chapter 4 describes the development of a new methodology for the determination of the absolute configuration of monosaccharide components of glycans. This methodology was used in the structural elucidation of the O-antigen PS of E. coli O155 (Chapter 5) that was carried out in a semi-automated manner using the program CASPER and unassigned NMR data. The conformational preferences of O-antigen PS of E. coli O5ac and O5ab are analyzed in Chapter 6, using a combination of NMR spectroscopy and molecular modeling methods. In Chapter 7 the structural analysis is focused on the core region of the LPS, and the structures of the deacylated lipooligosaccharides of three rough mutants of B. melitesis are reported. In several of the aforementioned chapters, the biosynthetic aspects behind the assembly of the respective PSs were examined on the bases of genetic information available in the NCBI and ECODAB databases.  Finally, in Chapter 8, different NMR pulse sequences available for the study of proteins and nucleic acids were evaluated and optimized for the structural analysis of 13C uniformly-labeled oligo- and polysaccharides.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2013. p. 83
Keyword
Nuclear Magnetic Resonance, carbohydrates, O-antigen polysaccharide
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-93833 (URN)978-91-7447-758-0 (ISBN)
Public defence
2013-10-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework Programme, 215536
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 6: Manuscript. Paper 7: Manuscript.

Available from: 2013-09-26 Created: 2013-09-17 Last updated: 2018-03-14Bibliographically approved

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