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Noncooperative folding of subdomains in Adenylate Kinase
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. (Gerhard Gröbner)
Visa övriga samt affilieringar
2009 (Engelska)Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 9, s. 1911-1927Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
ACS Publications , 2009. Vol. 48, nr 9, s. 1911-1927
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-18900DOI: 10.1021/bi8018042OAI: oai:DiVA.org:umu-18900DiVA, id: diva2:175006
Tillgänglig från: 2009-02-26 Skapad: 2009-02-26 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Ingår i avhandling
1. NMR studies of protein dynamics and structure
Öppna denna publikation i ny flik eller fönster >>NMR studies of protein dynamics and structure
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Enzymes are extraordinary molecules that can accelerate chemical reactions by several orders of magnitude. With recent advancements in structural biology together with classical enzymology the mechanism of many enzymes has become understood at the molecular level. During the last ten years significant efforts have been invested to understand the structure and dynamics of the actual catalyst (i. e. the enzyme). There has been a tremendous development in NMR spectroscopy (both hardware and pulse programs) that have enabled detailed studies of protein dynamics. In many cases there exists a strong coupling between enzyme dynamics and function. Here I have studied the conformational dynamics and thermodynamics of three model systems: adenylate kinase (Adk), Peroxiredoxin Q (PrxQ) and the structural protein S16. By developing a novel chemical shift-based method we show that Adk binds its two substrates AMP and ATP with an extraordinarily dynamic mechanism. For both substrate-saturated states the nucleotide-binding subdomains exchange between open and closed states, with the populations of these states being approximately equal. This finding contrasts with the traditional view of enzyme-substrate complexes as static low entropy states. We are also able to show that the individual subdomains in Adk fold and unfold in a non-cooperative manner. This finding is relevant from a functional perspective, since it allows a change in hydrogen bonding pattern upon substrate-binding without provoking global unfolding of the entire enzyme (as would be expected from a two-state folding mechanism). We also studied the structure and dynamics of the plant enzyme PrxQ in both reduced and oxidized states. Experimentally validated structural models were generated for both oxidation states. The reduced state displays unprecedented μs-ms conformational dynamics and we propose that this dynamics reflects local and functional unfolding of an α-helix in the active site. Finally, we solved the structure of S16 from Aquifex aeolicus and propose a model suggesting a link between thermostability and structure for a mesophilic and hyperthermophilic protein pair. A connection between the increased thermostability in the thermophilic S16 and residual structure in its unfolded state was discovered, persistent at high denaturant concentrations, thereby affecting the difference in heat capacity difference between the folded and unfolded state. In summary, we have contributed to the understanding of protein dynamics and to the coupling between dynamics and catalytic activity in enzymes.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet. Kemiska institutionen, 2010. s. 53
Nyckelord
NMR, protein dynamics, relaxation, protein folding
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci) Fysikalisk kemi
Forskningsämne
biokemi; fysikalisk kemi
Identifikatorer
urn:nbn:se:umu:diva-36790 (URN)978-91-7459-092-0 (ISBN)
Disputation
2010-11-05, KBC-huset, KB3A9, 901 87 Umeå, Umeå, 10:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2010-10-15 Skapad: 2010-10-11 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
2. Thermodynamical and structural properties of proteins and their role in food allergy
Öppna denna publikation i ny flik eller fönster >>Thermodynamical and structural properties of proteins and their role in food allergy
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2013. s. 33
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1571
Nyckelord
Protein folding, NMR, Food allergy, allergen, protein interactions
Nationell ämneskategori
Biofysik Strukturbiologi Biokemi och molekylärbiologi Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
medicinsk biokemi
Identifikatorer
urn:nbn:se:umu:diva-68020 (URN)978-91-7459-613-7 (ISBN)
Disputation
2013-05-03, KB3A9, plan 3, KBC-huset, Umeå Universitet, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2013-04-12 Skapad: 2013-04-10 Senast uppdaterad: 2018-06-08Bibliografiskt granskad

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