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Thermodynamical and structural properties of proteins and their role in food allergy
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Göran Larsson)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are important building blocks of all living organisms. They are composed of a defined sequence of different amino acids, and fold into a specific three-dimensional, ordered structure. The three-dimensional structure largely determines the function of the protein, but protein function always requires motion. Small movements within the protein structure govern the functional properties, and this thesis aims to better understand these discrete protein movements. The motions within the protein structure are governed by thermodynamics, which therefore is useful to predict protein interactions.

Nuclear magnetic resonance (NMR) is a powerful tool to study proteins at atomic resolution. Therefore, NMR is the primary method used within this thesis, along with other biophysical techniques such as Fluorescence spectroscopy, Circular Dichroism spectroscopy and in silico modeling.

In paper I, NMR in combination with molecular engineering is used to show that the folding of the catalytical subdomains of the enzyme Adenylate kinase does not affect the core of the protein, and thus takes a first step to linking folding, thermodynamic stability and catalysis.

In paper II, the structure of the primary allergen from Brazil nut, Ber e 1, is presented along with biophysical measurements that help explain the allergenic potential of the protein.

Paper III describes the need for a specific Brazil nut lipid fraction needed to induce an allergenic response. NMR and fluorescence spectroscopy is used to show that there is a direct interaction between Ber e 1 and one or several components in the lipid fraction.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2013. , 33 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1571
Keyword [en]
Protein folding, NMR, Food allergy, allergen, protein interactions
National Category
Biophysics Structural Biology Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Medical Biochemistry
URN: urn:nbn:se:umu:diva-68020ISBN: 978-91-7459-613-7OAI: diva2:615481
Public defence
2013-05-03, KB3A9, plan 3, KBC-huset, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2013-04-12 Created: 2013-04-10 Last updated: 2013-04-12Bibliographically approved
List of papers
1. Noncooperative folding of subdomains in Adenylate Kinase
Open this publication in new window or tab >>Noncooperative folding of subdomains in Adenylate Kinase
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 9, 1911-1927 p.Article in journal (Refereed) Published
Abstract [en]

Conformational change is regulating the biological activity of a large number of proteins and enzymes. Efforts in structural biology have provided molecular descriptions of the interactions that stabilize the stable ground states on the reaction trajectories during conformational change. Less is known about equilibrium thermodynamic stabilities of the polypeptide segments that participate in structural changes and whether the stabilities are relevant for the reaction pathway. Adenylate kinase (Adk) is composed of three subdomains: CORE, ATPlid, and AMPbd. ATPlid and AMPbd are flexible nucleotide binding subdomains where large-scale conformational changes are directly coupled to catalytic activity. In this report, the equilibrium thermodynamic stabilities of Adk from both mesophilic and hyperthermophilic bacteria were investigated using solution state NMR spectroscopy together with protein engineering experiments. Equilibrium hydrogen to deuterium exchange experiments indicate that the flexible subdomains are of significantly lower thermodynamic stability compared to the CORE subdomain. Using site-directed mutagenesis, parts of ATPlid and AMPbd could be selectively unfolded as a result of perturbation of hydrophobic clusters located in these respective subdomains. Analysis of the perturbed Adk variants using NMR spin relaxation and Cα chemical shifts shows that the CORE subdomain can fold independently of ATPlid and AMPbd; consequently, folding of the two flexible subdomains occurs independently of each other. Based on the experimental results it is apparent that the flexible subdomains fold into their native structure in a noncooperative manner with respect to the CORE subdomain. These results are discussed in light of the catalytically relevant conformational change of ATPlid and AMPbd.

Place, publisher, year, edition, pages
ACS Publications, 2009
National Category
Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-18900 (URN)10.1021/bi8018042 (DOI)
Available from: 2009-02-26 Created: 2009-02-26 Last updated: 2013-04-12Bibliographically approved
2. Solution structure, copper binding and backbone dynamics of recombinant Ber e 1: the major allergen from brazil nut
Open this publication in new window or tab >>Solution structure, copper binding and backbone dynamics of recombinant Ber e 1: the major allergen from brazil nut
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2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 10, e46435- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. METHODOLOGY/PRINCIPAL FINDINGS: The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using (15)N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu(2+) in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. CONCLUSIONS/SIGNIFICANCE: The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu(2+) ions. This Cu(2+) binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu(2+) can in turn affect the interaction that is essential to provoke an allergenic response.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2012
National Category
Chemical Sciences
urn:nbn:se:umu:diva-60707 (URN)10.1371/journal.pone.0046435 (DOI)000309580800019 ()23056307 (PubMedID)
Available from: 2012-10-29 Created: 2012-10-23 Last updated: 2013-08-09Bibliographically approved
3. Lipids are required for the development of Brazil nut allergy: the role of mouse and human iNKT cells
Open this publication in new window or tab >>Lipids are required for the development of Brazil nut allergy: the role of mouse and human iNKT cells
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2013 (English)In: Allergy. European Journal of Allergy and Clinical Immunology, ISSN 0105-4538, E-ISSN 1398-9995, Vol. 68, no 1, 74-83 p.Article in journal (Refereed) Published
Abstract [en]

Background Lipids are required for mice sensitization to Ber e 1, Brazil nut major allergen. Here, we characterized different lipid fractions extracted from Brazil nuts and the lipid-binding ability of Ber e 1. Further, we determined their in vivo ability to induce Ber-specific anaphylactic antibodies and the role of invariant natural killer T (iNKT) cells in this process.

Methods Wild-type (WT) and iNKT cell-deficient mice were sensitized with Ber e 1 and specific lipid fractions, and anaphylactic antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA). The lipid-binding characteristic of Ber e 1 (Ber) was established by using fluorescent probes and 15N-labeled NMR. In vitro production of IL-4 was determined in Ber/lipid C-stimulated mouse iNKT cells and human T-cell lines containing NKTs primed with CD1d+C1R transfectants by flow cytometry and ELISA, respectively.

Results Only one specific lipid fraction (lipid C), containing neutral and common phospholipids, induced Ber anaphylactic antibodies in mice. Ber e 1 has a lipid-binding site, and our results indicated an interaction between Ber e 1 and lipid C. iNKT-deficient mice produced lower levels of anaphylactic antibodies than WT mice. In vitro, Ber/lipid C-stimulated murine iNKT cells produced IL-4 but not IFN-gamma. Human T-cell lines derived from nut-allergic patients produced IL-4 to Ber/lipid C in a CD1d- and dose-dependent manner.

Conclusion Lipid fraction C from Brazil nut presents an essential adjuvant activity to Ber e 1 sensitization, and iNKT cells play a critical role in the development of Brazil nut-allergic response.

Place, publisher, year, edition, pages
Hoboken, NJ, USA: Wiley-Blackwell, 2013
anaphylactic antibodies, food allergy, humans, lipids, NKT cells
National Category
Respiratory Medicine and Allergy Immunology in the medical area
urn:nbn:se:umu:diva-63751 (URN)10.1111/all.12057 (DOI)000311974500009 ()
Available from: 2013-01-10 Created: 2013-01-07 Last updated: 2013-04-12Bibliographically approved

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