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Structural Studies of Flexible Biomolecules and a DNA-binding Protein
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Pål Stenmark)
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 [en]
Maximum entropy, motilin, DNA-binding proteins, Karplus equation, disaccaride, direct repeats
National Category
Structural Biology
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-42009ISBN: 978-91-7447-102-1 (print)OAI: oai:DiVA.org:su-42009DiVA: diva2:343499
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
List of papers
1. Maximum entropy reconstruction of joint phi, psi-distribution with a coil-library prior: the backbone conformation of the peptide hormone motilin in aqueous solution from phi and psi-dependent J-couplings
Open this publication in new window or tab >>Maximum entropy reconstruction of joint phi, psi-distribution with a coil-library prior: the backbone conformation of the peptide hormone motilin in aqueous solution from phi and psi-dependent J-couplings
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2007 (English)In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 38, no 2, 107-23 p.Article in journal (Refereed) Published
Keyword
Circular Dichroism, Entropy, Humans, Motilin/*chemistry, Nuclear Magnetic Resonance; Biomolecular/*methods, Protein Structure; Secondary, Solutions
Identifiers
urn:nbn:se:su:diva-21324 (URN)10.1007/s10858-007-9150-1 (DOI)000246613100001 ()17458509 (PubMedID)
Available from: 2007-12-08 Created: 2007-12-08 Last updated: 2010-08-13Bibliographically approved
2. 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
Open this publication in new window or tab >>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
Show others...
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.

Keyword
Maximum entropy, Karplus relation, disaccharides
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-42005 (URN)10.1039/c003958f (DOI)000280527500015 ()
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically approved
3. Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein
Open this publication in new window or tab >>Assignment of 1H, 13C, and 15N chemical shift resonances of P2 C-repressor protein
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2008 (Swedish)In: Biomolecular NMR Assignments, ISSN 1874-270X, Vol. 2, no 2, 215-217 p.Article in journal (Refereed) Published
Keyword
P2, Bacteriophage, Repressor, HTH, Homodimer
Identifiers
urn:nbn:se:su:diva-14874 (URN)10.1007/s12104-008-9124-6 (DOI)000260614600031 ()
Available from: 2009-01-12 Created: 2009-01-12 Last updated: 2010-08-13Bibliographically approved
4. Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats
Open this publication in new window or tab >>Crystal structure of the P2 C-repressor: a binder of nonpalindromic direct DNA repeats
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2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 21, 7778-7790 p.Article in journal (Refereed) Published
Abstract [en]

As opposed to the vast majority of prokaryoticrepressors, the immunity repressor of temperateEscherichia coli phage P2 (C) recognizes nonpalindromicdirect repeats of DNA rather thaninverted repeats. We have determined the crystalstructure of P2 C at 1.8A ° . This constitutes the firststructure solved from the family of C proteins fromP2-like bacteriophages. The structure reveals thatthe P2 C protein forms a symmetric dimer orientedto bind the major groove of two consecutive turns ofthe DNA. Surprisingly, P2 C has great similarities tobinders of palindromic sequences. Nevertheless, thetwo identical DNA-binding helixes of the symmetricP2 C dimer have to bind different DNA sequences.Helix 3 is identified as the DNA-recognition motif inP2 C by alanine scanning and the importance for theindividual residues in DNA recognition is defined.A truncation mutant shows that the disorderedC-terminus is dispensable for repressor function.The short distance between the DNA-bindinghelices together with a possible interaction betweentwo P2 C dimers are proposed to be responsible forextensive bending of the DNA. The structure providesinsight into the mechanisms behind the mutants ofP2 C causing dimer disruption, temperature sensitivityand insensitivity to the P4 antirepressor.

Keyword
DNA-binding protein, direct repeats, P2 C repressor
National Category
Structural Biology
Research subject
Structural Biology; Biochemistry
Identifiers
urn:nbn:se:su:diva-42003 (URN)10.1093/nar/gkq626 (DOI)000284952000042 ()
Funder
The Wenner-Gren FoundationSwedish Foundation for Strategic Research Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically approved
5. NMR Structure Note: The C Repressor of the P2 Bacteriophage
Open this publication in new window or tab >>NMR Structure Note: The C Repressor of the P2 Bacteriophage
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(English)Manuscript (preprint) (Other academic)
Keyword
P2 C repressor, DNA-binding protein, direct repeats
Identifiers
urn:nbn:se:su:diva-42016 (URN)
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2010-08-14Bibliographically approved

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