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NMR studies of protein dynamics and structure
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Magnus Wolf-Watz)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Umeå: Umeå universitet. Kemiska institutionen , 2010. , 53 p.
Keyword [en]
NMR, protein dynamics, relaxation, protein folding
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Physical Chemistry
Research subject
Biochemistry; Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-36790ISBN: 978-91-7459-092-0 (print)OAI: oai:DiVA.org:umu-36790DiVA: diva2:356223
Public defence
2010-11-05, KBC-huset, KB3A9, 901 87 Umeå, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2010-10-15 Created: 2010-10-11 Last updated: 2010-10-15Bibliographically approved
List of papers
1. NMR identification of transient complexes critical to adenylate kinase catalysis
Open this publication in new window or tab >>NMR identification of transient complexes critical to adenylate kinase catalysis
2007 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 45, 14003-12 p.Article in journal (Refereed) Published
Abstract [en]

A fundamental question in protein chemistry is how the native energy landscape of enzymes enables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studies have revealed that substrate binding is accompanied by large rate-limiting spatial displacements of both the ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe the native energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and in the presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATP binding motif populates both the open and the closed conformations with almost equal populations. A similar scenario is observed for AMP binding, which induces an equilibrium between open and closed conformations of the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes that exist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substrate binding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic coupling between the ATP and AMP binding sites was discovered. On the basis of these and previous results, we propose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existing equilibria toward catalytically active states.

Identifiers
urn:nbn:se:umu:diva-16865 (URN)doi:10.1021/ja075055g (DOI)17935333 (PubMedID)
Note
Web Release Date: October 13, 2007Available from: 2007-12-11 Created: 2007-12-11 Last updated: 2017-12-14Bibliographically approved
2. 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
Identifiers
urn:nbn:se:umu:diva-18900 (URN)10.1021/bi8018042 (DOI)
Available from: 2009-02-26 Created: 2009-02-26 Last updated: 2017-12-13Bibliographically approved
3. Arabidopsis thaliana peroxiredoxin Q is extraordinarily dynamic on the μs-ms timescale
Open this publication in new window or tab >>Arabidopsis thaliana peroxiredoxin Q is extraordinarily dynamic on the μs-ms timescale
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Peroxiredoxin Q (PrxQ) isolated from Arabidopsis thaliana belongs to a family of redox enzymes called peroxiredoxins, which are thioredoxin- or glutaredoxin dependent peroxidases acting to reduce peroxides and in particular hydrogen peroxide. PrxQ cycles between an active reduced state and an inactive oxidized state during its catalytic cycle. The catalytic mechanism involves a nucleophilic attack of the catalytic cysteine on hydrogen peroxide to generate a sulfonic acid intermediate with a concerted release of a water molecule. This intermediate is subsequently relaxed by the reaction of a second cysteine, denoted as the resolving cysteine, generating an intermolecular disulphide bond to expel a second water molecule into solution. PrxQ is finally recycled to the active state by a thioredoxin dependent reduction. Previous structural studies of PrxQ homologues have provided the structural basis for the switch between reduced and oxidized conformations. Here we have performed a detailed study of the structure and dynamics of PrxQ in both the oxidized and reduced state. Reliable and experimentally validated structural models of PrxQ in both oxidation states were generated using homology based modeling. Model-free analyses of NMR spin relaxation show that PrxQ is monomeric in both oxidation states. As evident from fast R2 relaxation rates the reduced form of PrxQ undergoes unprecedented dynamics on the slow μs-ms timescale. The ground state of the conformational dynamics is likely the stably folded reduced state as implied by circular dichroism spectroscopy. We speculate that the extensive dynamics is intimately related to the catalytic function of PrxQ.

Keyword
activity, circular dichroism, dynamics, enzyme, NMR
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biophysics
Research subject
Biochemistry; Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-36751 (URN)
Available from: 2010-10-11 Created: 2010-10-11 Last updated: 2010-10-15Bibliographically approved
4. Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state
Open this publication in new window or tab >>Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 4, 845-858 p.Article in journal (Refereed) Published
Abstract [en]

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16meso) and hyperthermophilic (S16thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔCp0) of 6.4 and 3.3 kJ mol− 1 K− 1, respectively. A strong linear correlation between ΔCp0 and melting temperature (Tm) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16meso folds through an apparent two-state model, whereas S16thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16thermo is more compact relative to S16meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16thermo that appears to account for the large difference in quantified ΔCp0 values and, in turn, parts of the observed extreme thermal stability of S16thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.

Keyword
ribosomal protein S16, residual structure, protein folding, thermostability, ΔCp0
Identifiers
urn:nbn:se:umu:diva-9439 (URN)10.1016/j.jmb.2008.04.007 (DOI)
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2017-12-14Bibliographically approved

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