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Population distribution of flexible molecules from maximum entropy analysisusing different priors as background information: application to the phi,psi-conformational space of the a-(1→2)-linked mannose disaccharide presentin N- and O-linked glycoproteins
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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2010 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 8, no 16, 3684-3695 p.Article in journal (Refereed) Published
Abstract [en]

The conformational space available to the flexible molecule a-D-Manp-(1→2)-a-D-Manp-OMe, amodel for the a-(1→2)-linked mannose disaccharide in N- or O-linked glycoproteins, is determinedusing experimental data and molecular simulation combined with a maximum entropy approach thatleads to a converged population distribution utilizing different input information. A database survey ofthe Protein Data Bank where structures having the constituent disaccharide were retrieved resulted inan ensemble with >200 structures. Subsequent filtering removed erroneous structures and gave thedatabase (DB) ensemble having three classes of mannose-containing compounds, viz., N- and O-linkedstructures, and ligands to proteins. A molecular dynamics (MD) simulation of the disaccharide revealeda two-state equilibrium with a major and a minor conformational state, i.e., the MD ensemble. Thesetwo different conformation ensembles of the disaccharide were compared to measured experimentalspectroscopic data for the molecule in water solution. However, neither of the two populations werecompatible with experimental data from optical rotation, NMR 1H,1H cross-relaxation rates as well ashomo- and heteronuclear 3J couplings. The conformational distributions were subsequently used asbackground information to generate priors that were used in a maximum entropy analysis. Theresulting posteriors, i.e., the population distributions after the application of the maximum entropyanalysis, still showed notable deviations that were not anticipated based on the prior information.Therefore, reparameterization of homo- and heteronuclear Karplus relationships for the glycosidictorsion angles f and y were carried out in which the importance of electronegative substituents on thecoupling pathway was deemed essential resulting in four derived equations, two 3JCOCC and two 3JCOCHbeing different for the f and y torsions, respectively. These Karplus relationships are denotedJCX/SU09. Reapplication of the maximum entropy analysis gave excellent agreement between theMD- and DB-posteriors. The information entropies show that the current reparametrization of theKarplus relationships constitutes a significant improvement. The fH torsion angle of the disaccharide isgoverned by the exo-anomeric effect and for the dominating conformation fH = -40◦ and yH = 33◦.The minor conformational state has a negative yH torsion angle; the relative populations of the majorand the minor states are ~3 : 1. It is anticipated that application of the methodology will be useful toflexible molecules ranging from small organic molecules to large biomolecules.

Place, publisher, year, edition, pages
2010. Vol. 8, no 16, 3684-3695 p.
Keyword [en]
Maximum entropy, Karplus relation, disaccharides
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-42005DOI: 10.1039/c003958fISI: 000280527500015OAI: oai:DiVA.org:su-42005DiVA: diva2:343486
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Structural Studies of Flexible Biomolecules and a DNA-binding Protein
Open this publication in new window or tab >>Structural Studies of Flexible Biomolecules and a DNA-binding Protein
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The knowledge of the three-dimensional structures of proteins and polypeptides is essential to understand their functions. The work shown in this thesis has two objectives. The first one is to develop a new analytical method based on maximum entropy (ME) theory to analyze NMR experimental data such as NOEs and J-couplings in order to reconstitute φ,ψ Ramachandran plots of flexible biomolecules. Two model systems have been used, the flexible polypeptide motilin and the disaccharide α-D-Mannosep-(1-2)-α-D-Mannosep-O-Me (M2M). The experimental data was defined as constraints that were combined with prior information (priors) which were the φ,ψ distributions obtained from either a coil library, the Protein DataBank or Molecular Dynamics Simulations. ME theory was utilized to formulate φ,ψ distributions (posteriors) that are least committed to the priors and in full agreement with the experimental data. Reparamerization of the Karplus relation was necessary to obtain realistic distributions for the M2M. Clear structural propensities were found in motilin with a nascent α-helix in the central part (residues Y7-E17), a left handed 31 helix in the C-terminus (R18-G21) and an extended conformation in the N-terminus. The contribution of each residue to the thermodynamic entropy (segmental entropy) was calculated from the posteriors and compared favorably to the segmental entropies estimated from 15N-relaxation data. For M2M the dominating conformation of the glycosidic linkage was found to be at φH=-40° ψH=33°, which is governed by the exo-anomeric effect. Another minor conformation with a negative ψH angle was discovered in M2M. The ratio between both populations is about 3:1. The second part of the thesis is a structural study of a DNA-binding protein, the C repressor of the P2 bacteriophage (P2 C). P2 C represses the lytic genes of the P2 bacteriophage, thereby directing the P2 lifecycle toward the lysogenic lifemode. The crystal and solution structures of P2 C have been solved by X-ray crystallography and NMR, respectively. Both structures revealed a homodimeric protein with five rigid α-helices made up by residues 5-66 and a β-strand conformation in residues 69-76 in each monomer. 15N-relaxation data showed that the C-terminus (residues 85-99) is highly flexible and fully unstructured. A model representing the P2 C-DNA complex was built based on the structure and available biochemical data. In the model, P2 C binds DNA cooperatively and two homodimeric P2 C molecules are close enough to interact and bind one direct DNA repeat each.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 76 p.
Keyword
Maximum entropy, motilin, DNA-binding proteins, Karplus equation, disaccaride, direct repeats
National Category
Structural Biology
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-42009 (URN)978-91-7447-102-1 (ISBN)
Public defence
2010-09-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 09:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: In press. Paper 5: Manuscript. Available from: 2010-08-23 Created: 2010-08-13 Last updated: 2010-08-17Bibliographically approved
2. NMR spectroscopy and MD simulations of carbohydrates
Open this publication in new window or tab >>NMR spectroscopy and MD simulations of carbohydrates
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Knowledge about the structure, conformation and dynamics of carbohydrates is important in our understanding of the way carbohydrates function in biological systems, for example in intermolecular signaling and recognition. This thesis is a summary of five papers studying these properties in carbohydrate-containing molecules with NMR spectroscopy and molecular dynamics simulations.

In paper I, the ring-conformations of the six-membered rings of two carbaiduronic analogs were investigated. These carbasugars could potentially be used as hydrolytically stable mimics of iduronic acid in drugs. The study showed that the equilibrium is entirely shifted towards the 4C1 conformation.

Paper II is an investigation of the conformational flexibility and dynamics of two (1→6)-linked disaccharides related to an oligosaccharide epitope expressed on malignant tumor cells.

In paper III, the conformational space of the glycosidic linkage of an alfa-(1→2) linked mannose disaccharide present in N- and O-linked glycoproteins, was studied. A maximum entropy analysis using different priors as background information was used and four new Karplus equations for 3JC,C and 3JC,H coupling constants, related to the glycosidic linkage, were presented.

Paper IV describes a structural elucidation of the exopolysaccharide (EPS) produced by Streptococcus thermophilus ST1, a major dairy starter used in yoghurt and cheese production. The EPS contains a hexasaccharide repeating unit of d-galactose and d-glucose residues, which is a new EPS structure of the S. thermophilus species.

In paper V, the dynamics of three generations of glycodendrimers were investigated by NMR diffusion and 13C NMR relaxation studies. Three different correlations times were identified, one global correlation time describing the rotation of the dendrimer as a whole, one local correlation time describing the reorientation of the C-H vectors, and one correlation time describing the pulsation of a dendrimer branch.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2011. 75 p.
Keyword
NMR specroscopy, MD simulations, carbohydrates
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-61569 (URN)978-91-7447-348-3 (ISBN)
Public defence
2011-09-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2011-09-08 Created: 2011-08-23 Last updated: 2011-08-23Bibliographically approved

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