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NMR spectroscopy in structural and conformational analysis of bacterial polysaccharides
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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. , 83 p.
Keyword [en]
Nuclear Magnetic Resonance, carbohydrates, O-antigen polysaccharide
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-93833ISBN: 978-91-7447-758-0 (print)OAI: oai:DiVA.org:su-93833DiVA: diva2:649204
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: 2013-10-08Bibliographically approved
List of papers
1. Structural studies and biosynthetic aspects of the o antigen polysaccharide from Escherichia coli o174
Open this publication in new window or tab >>Structural studies and biosynthetic aspects of the o antigen polysaccharide from Escherichia coli o174
2012 (English)In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 354, 102-105 p.Article in journal (Refereed) Published
Abstract [en]

The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with H-1 and C-13 NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by H-1, C-13-heteronuclear multiple-bond correlation and H-1, H-1-NOESY experiments. The PS is composed of tetrasaccharide repeating units with the following structure: -> 4)-beta-D-GlcpA-(1 -> 3)-beta-D-Galp-(1 -> 3)-beta-D-GalpNAc-(1 -> vertical bar beta-D-GlcpNAc-(1 -> 2) Cross-peaks of low intensity were present in the NMR spectra consistent with a beta-D-GlcpNAc-(1 -> 2)-beta-D-GlcpA(1 -> structural element at the terminal part of the polysaccharide, which on average is composed of similar to 15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.

Keyword
Escherichia coli, Lipopolysaccharide, NMR, Biological repeating unit
National Category
Organic Chemistry Biochemistry and Molecular Biology
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-79797 (URN)10.1016/j.carres.2012.02.020 (DOI)000304055100012 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:4;

Available from: 2012-09-11 Created: 2012-09-11 Last updated: 2017-12-07Bibliographically approved
2. Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof
Open this publication in new window or tab >>Structural studies of the O-antigen polysaccharide from Escherichia coli O115 and biosynthetic aspects thereof
2013 (English)In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 23, no 3, 354-362 p.Article in journal (Refereed) Published
Abstract [en]

The structure of the O-antigen polysaccharide (PS) of Escherichia coliO115 has been investigated using a combination of component analysis and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy experiments. The repeating unit of the O-antigen was elucidated using the O-deacetylated PS and has the following branched pentasaccharide structure: →3)[β-L-Rhap-(1 → 4)]-β-D-GlcpNAc-(1 → 4)-α-D-GalpA-(1 → 3)-α-D-Manp-(1 → 3)-β-D-GlcpNAc-(1→. Cross-peaks of low intensity, corresponding to a β-L-Rhap-(1 → 4)-β-D-GlcpNAc-(1→ structural element, were present in the NMR spectra and attributed to the terminal part of the PS; this information defines the biological repeating unit of the O-antigen by having a 3-substituted N-acetyl-D-glucosamine (GlcNAc) residue at its reducing end. Analysis of the NMR spectra of the native PS revealed O-acetyl groups distributed over different positions of theL-Rhap residue (∼0.70 per repeating unit) as well as at O-2 and O-3 of the D-GalpA residue (∼0.03 and ∼0.25 per repeating unit, respectively), which is in agreement with the presence of two acetyltransferases previously identified in the O-antigen gene cluster (Wang Q, Ruan X, Wei D, Hu Z, Wu L, Yu T, Feng L, Wang L. 2010. Mol Cell Probes. 24:286–290.). In addition, the four glycosyltransferases initially identified in the O-antigen gene cluster of E. coli O115 were analyzed using BLAST, and the function of two of them predicted on the basis of similarities with glycosyltransferases from Shigella dysenteriae type 5 and 12, as well as E. coli O58 and O152.

Keyword
Escherichia coli, glycosyltransferases, lipopolysaccharide, O-acetylation, O-antigen
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-93837 (URN)10.1093/glycob/cws161 (DOI)
Funder
EU, FP7, Seventh Framework Programme, 215536Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2013-09-17 Created: 2013-09-17 Last updated: 2017-12-06Bibliographically approved
3. Automatic structure determination of regular polysaccharides based solely on NMR spectroscopy
Open this publication in new window or tab >>Automatic structure determination of regular polysaccharides based solely on NMR spectroscopy
2011 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 11, 3851-3855 p.Article in journal (Refereed) Published
Abstract [en]

The structural analysis of polysaccharides requires that the sugar components and their absolute configurations are determined. We here show that this can be performed based on NMR spectroscopy by utilizing butanolysis with (+)- and (-)-2-butanol that gives the corresponding 2-butyl glycosides with characteristic 1H and 13C NMR chemical shifts. The subsequent computer-assisted structural determination by CASPER can then be based solely on NMR data in a fully automatic way as shown and implemented herein. The method is additionally advantageous in that reference data only have to be prepared once and from a user's point of view only the unknown sample has to be derivatized for use in CASPER.

National Category
Organic Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-70868 (URN)10.1021/bm201169y (DOI)000296830600003 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

authorCount :3

Available from: 2012-01-25 Created: 2012-01-24 Last updated: 2017-12-08Bibliographically approved
4. Facile Structural Elucidation of Glycans Using NMR Spectroscopy Data and the Program CASPER: Application to the O-Antigen Polysaccharide of Escherichia coli O155
Open this publication in new window or tab >>Facile Structural Elucidation of Glycans Using NMR Spectroscopy Data and the Program CASPER: Application to the O-Antigen Polysaccharide of Escherichia coli O155
2013 (English)In: ChemPlusChem, ISSN 2192-6506, Vol. 78, no 11, 1327-1329 p.Article in journal (Refereed) Published
Abstract [en]

The program CASPER was successfully employed to rapidly elucidate a new O-antigen polysaccharide structure (obtained from a strain of Escherichia coli serogroup O155), using solelyunassigned NMR spectroscopy data as input information. Thus, what is considered the most tedious and time-consuming part of the structural elucidation process has been reduced from several hours (or even days) of manual interpretation to about four minutes of automated analysis.

Keyword
analytical methods, antigens, carbohydrates, CASPER, NMR spectroscopy
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-93838 (URN)10.1002/cplu.201300273 (DOI)000326900600005 ()
Funder
EU, FP7, Seventh Framework Programme, 215536Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2013-09-17 Created: 2013-09-17 Last updated: 2016-10-18Bibliographically approved
5. Conformational Preferences of the O-Antigen Polysaccharides of Escherichia coli O5ac and O5ab Using NMR Spectroscopy and Molecular Modeling
Open this publication in new window or tab >>Conformational Preferences of the O-Antigen Polysaccharides of Escherichia coli O5ac and O5ab Using NMR Spectroscopy and Molecular Modeling
Show others...
2013 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 7, 2215-2224 p.Article in journal (Refereed) Published
Abstract [en]

Escherichia coli serogroup O5 comprises two different subgroups (O5ab and O5ac), which are indiscernible from the point of view of standard immunological serotyping. The structural similarities between the O-antigen polysaccharides (PSs) of these two strains are remarkable, with the only difference being the glycosidic linkage connecting the biological tetrasaccharide repeating units. In the present study, a combination of NMR spectroscopy and molecular modeling methods were used to elucidate the conformational preferences of these two PSs. The NMR study was based on the analysis of intra- and inter-residue proton-proton distances using NOE build-up curves. Molecular models of the repeating units and their extension to polysaccharides were obtained, taking into account the conformational flexibility as assessed by the force field applied and a genetic algorithm. The agreements between experimentally measured and calculated distances could only be obtained by considering an averaging of several low energy conformations observed in the molecular models.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-92630 (URN)10.1021/bm400354y (DOI)000321793700011 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme
Note

AuthorCount:5;

Available from: 2013-08-19 Created: 2013-08-14 Last updated: 2017-12-06Bibliographically approved
6. Structural studies of the rough lipopolysaccharides of Brucella melitensis mutants affected in the biosynthesis of the core and O-antigen polysaccharide
Open this publication in new window or tab >>Structural studies of the rough lipopolysaccharides of Brucella melitensis mutants affected in the biosynthesis of the core and O-antigen polysaccharide
(English)Manuscript (preprint) (Other academic)
Keyword
Brucella melitensis, core, Nuclear Magnetic Resonance, carbohydrates
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-93839 (URN)
Funder
EU, FP7, Seventh Framework Programme, 215536
Available from: 2013-09-17 Created: 2013-09-17 Last updated: 2013-09-19Bibliographically approved
7. 13C-based NMR spectroscopy for Structure Analysis of Uniformly 13C-labeled Oligo- and Polysaccharides
Open this publication in new window or tab >>13C-based NMR spectroscopy for Structure Analysis of Uniformly 13C-labeled Oligo- and Polysaccharides
(English)Manuscript (preprint) (Other academic)
Keyword
Nuclear Magnetic Resonance, 13C-labelling, carbohydrates
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-93840 (URN)
Funder
EU, European Research Council, 215536
Available from: 2013-09-17 Created: 2013-09-17 Last updated: 2013-09-19Bibliographically approved

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