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Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Gerhard Gröbner)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology, 44202 Dortmund, Germany.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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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.

Place, publisher, year, edition, pages
2008. Vol. 379, no 4, 845-858 p.
Keyword [en]
ribosomal protein S16, residual structure, protein folding, thermostability, ΔCp0
Identifiers
URN: urn:nbn:se:umu:diva-9439DOI: 10.1016/j.jmb.2008.04.007OAI: oai:DiVA.org:umu-9439DiVA: diva2:149110
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2014-03-11Bibliographically approved
In thesis
1. NMR studies of protein dynamics and structure
Open this publication in new window or tab >>NMR studies of protein dynamics and structure
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
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:nbn:se:umu:diva-36790 (URN)978-91-7459-092-0 (ISBN)
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
2. Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
Open this publication in new window or tab >>Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions.

The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso.

The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding.

We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur.

In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence. 

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2014. 55 p.
Keyword
Fluorescence, electronic energy transfer, two-photon excitation, small ribosomal protein S16, macromolecular crowding, dextran 20
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-86794 (URN)978-91-7601-021-1 (ISBN)
Public defence
2014-04-04, KBC-huset, KB3B1, Umeå universitet, Linnaeus Väg, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 550021104
Available from: 2014-03-12 Created: 2014-03-11 Last updated: 2014-03-11Bibliographically approved

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Wallgren, MarcusÅdén, JörgenMikaelsson, ThereseJohansson, Lennart B-ÅWolf-Watz, Magnus

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