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Combining weak affinity chromatography, NMR spectroscopy and molecular simulations in carbohydrate-lysozyme interaction studies
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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2012 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 10, no 15, 3019-3032 p.Article in journal (Refereed) Published
Abstract [en]

By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using H-1 saturation transfer difference (STD) effects and transferred H-1,H-1-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides beta-D-Galp-(1 -> 4)-beta-D-GlcpNAc-OMe and beta-D-GlcpNAc-(1 -> 4)-beta-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/pi-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on H-1 NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.

Place, publisher, year, edition, pages
2012. Vol. 10, no 15, 3019-3032 p.
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-85915DOI: 10.1039/c2ob07066aISI: 000301958100017OAI: oai:DiVA.org:su-85915DiVA: diva2:585474
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2013-01-10 Created: 2013-01-10 Last updated: 2017-12-06Bibliographically approved
In thesis
1. The sweet side of molecular structure: NMR spectroscopic studies of glycans and their interactions with proteins
Open this publication in new window or tab >>The sweet side of molecular structure: NMR spectroscopic studies of glycans and their interactions with proteins
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis, within the topic of bioorganic chemistry, the molecular structure of carbohydrates has been studied. Carbohydrates, or glycans, are ubiquitous biomolecules exhibiting a wide range of biological roles. The specific functions of these molecules are largely determined by their interactions with proteins and molecular structure ultimately governs such specialized recognition events.

Glycan-binding proteins, such as lectins or enzymes, often interact with their sweet ligands in a transient fashion and nuclear magnetic resonance spectroscopy (NMR) is a viable technique to probe these complexes. In particular, ligand-based NMR techniques have been employed, typically in combination with other biophysical as well as biochemical and computational methods. The aim of this work has been to gain new insights about specific biological systems, to develop methods and to devise protocols for their studies.

The first two papers cover NMR-interaction studies of native ligands as well as inhibitor glycans with the enzyme hen egg-white lysozyme and the lectin botulinum neurotoxin type A. Screening experiments were performed to investigate ligand affinities and selectivities. Solution models in combination with X-ray crystal structures were compared in order to evaluate their agreement and the details of interactions.

A method for application in carbohydrate ligand NMR-screening was developed in paper three. The heteronucleus selenium was exploited as a reporter of selenoglycosides binding to lectins. 77Se NMR spectroscopy proved sensitive to binding events and the presented approach should be useful in large screenings of glycomimetic inhibitors.  In order to obtain sufficient amounts of glycans for bioorganic studies their production often relies on chemical synthesis. In the last paper, the structure of some conformationally highly activated glycosyl donors was thoroughly investigated and related to their reactivity in synthetic glycosylation reactions.  

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2015. 77 p.
Keyword
bioorganic chemistry, glycans, ligand-based NMR, molecular simulations, protein-carbohydrate interactions, lectins, carbohydrate conformations, 77Se NMR, structure-reactivity relationships, super-armed donors
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-112350 (URN)978-91-7649-083-9 (ISBN)
Public defence
2015-02-20, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

 

Available from: 2015-01-29 Created: 2015-01-12 Last updated: 2017-10-11Bibliographically approved

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