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On the mechanism of Urea-induced protein denaturation
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is well known that folded proteins in water are destabilized by the addition of urea. When a protein loses its ability to perform its biological activity due to a change in its structure, it is said to denaturate. The mechanism by which urea denatures proteins has been thoroughly studied in the past but no proposed mechanism has yet been widely accepted. The topic of this thesis is the study of the mechanism of urea-induced protein denaturation, by means of Molecular Dynamics (MD) computer simulations and Nuclear Magnetic Resonance (NMR) spectroscopy.

Paper I takes a thermodynamic approach to the analysis of protein – urea solution MD simulations. It is shown that the protein – solvent interaction energies decrease significantly upon the addition of urea. This is the result of a decrease in the Lennard-Jones energies, which is the MD simulation equivalent to van der Waals interactions. This effect will favor the unfolded protein state due to its higher number of protein - solvent contacts. In Paper II, we show that a combination of NMR spin relaxation experiments and MD simulations can successfully be used to study urea in the protein solvation shell. The urea molecule was found to be dynamic, which indicates that no specific binding sites exist. In contrast to the thermodynamic approach in Paper I, in Paper III we utilize MD simulations to analyze the affect of urea on the kinetics of local processes in proteins. Urea is found to passively unfold proteins by decreasing the refolding rate of local parts of the protein that have unfolded by thermal fluctuations.

Based upon the results of Paper I – III and previous studies in the field, I propose a mechanism in which urea denatures proteins mainly by an enthalpic driving force due to attractive van der Waals interactions. Urea interacts favorably with all the different parts of the protein. The greater solvent accessibility of the unfolded protein is ultimately the factor that causes unfolded protein structures to be favored in concentrated urea solutions.

Place, publisher, year, edition, pages
Umeå: Department of Chemistry, Umeå university , 2010. , 52 p.
Keyword [en]
Chemical denaturation, Protein unfolding, urea, MD simulations, NMR spectroscopy
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-33151ISBN: 978-91-7264-997-2 (print)OAI: oai:DiVA.org:umu-33151DiVA: diva2:310217
Public defence
2010-05-07, KBC huset, KB3A9, Umeå Universitet, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-16 Created: 2010-04-13 Last updated: 2010-09-24Bibliographically approved
List of papers
1. On the stability of chymotrypsin inhibitor 2 in a 10 M urea solution: the role of interaction energies for urea-induced protein denaturation
Open this publication in new window or tab >>On the stability of chymotrypsin inhibitor 2 in a 10 M urea solution: the role of interaction energies for urea-induced protein denaturation
2010 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 12, no 32, 9358-9366 p.Article in journal (Refereed) Published
Abstract [en]

Molecular dynamics simulations of chymotrypsin inhibitor 2 in both water and in 10 M urea have been compared with respect to the energies of interaction between protein and solvent. The analysis yield clear and detailed information regarding the enthalpic driving force of urea-induced protein denaturation. The protein is kept in the folded structure by applying positional restraints on the alpha-carbons, thereby creating an equilibrium system from which appropriate driving forces for denaturation can be obtained. All protein atoms are classified as belonging to the backbone, the polar side chains or to the hydrophobic side chains. The interaction energies are extracted for each class separately. The commonly proposed mechanisms of urea denaturation, i.e. that urea interacts mainly with the backbone or with the hydrophobic side chains, can then be tested. The results show that urea decreases the Lennard-Jones interaction energies between protein and solvent by a large amount. The electrostatic energies are almost unaffected by the switch of solvent. The energetically favorable interaction between CI2 and the urea solvent will function as a driving force for the protein to increase its solvent accessible surface area as compared to the folded protein in water. The magnitude of the decrease in the Lennard-Jones energies for the hydrophobic and the hydrophilic side chains and for the backbone were similar. We therefore conclude that urea interacts favorably with the whole protein surface and that all parts of the protein are important in urea-induced denaturation.

Place, publisher, year, edition, pages
Cambridge: RSC publishing, 2010
Keyword
urea, chemical denaturation, protein unfolding, md simulation, molecular dynamics simulation
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-35289 (URN)10.1039/b925726h (DOI)000280708400028 ()20563326 (PubMedID)
Available from: 2010-08-11 Created: 2010-08-11 Last updated: 2010-09-24Bibliographically approved
2. A combined molecular dynamic simulation and Urea 14N NMR relaxation study of the Urea - lysozyme system
Open this publication in new window or tab >>A combined molecular dynamic simulation and Urea 14N NMR relaxation study of the Urea - lysozyme system
2010 (English)In: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, Vol. 75, no 3, 953-9 p.Article in journal (Refereed) Published
Abstract [en]

Urea in the lysozyme solvation shell has been studied by utilizing a combination of urea , water NMR relaxation experiments and a molecular dynamics simulation of the urea–lysozyme system. Samples with lysozyme in the native fold in water as well as in 3 M urea have been studied, as well as denatured lysozyme in a 8.5 M urea solvent. The spin relaxation rates of the samples with folded protein show a clear field dependence, which is consistent with a few urea molecules having long residence times on the protein surface and preferentially located in pockets and grooves on the protein. By combining the 3 M urea NMR relaxation data and data from the MD simulation, a full parameter set of the relaxation model is found which can successfully predict the experimental relaxation rates of the 3 M urea sample. However, in the parameter fitting it is evident that the rotational dynamics of urea in the MD simulation is slightly too fast to be consistent with the NMR relaxation rates, perhaps a result of the fast dynamics of the TIP3P water model. The relaxation rates of urea in the proximity of the unfolded lysozyme lack field dependence, which can be interpreted as a loss of pockets and grooves on the denatured protein. The extracted model parameters from the 3 M sample are adjusted and tested on a simple model of the unfolded protein sample and are seen to be in agreement with the relaxation rates. It is shown that the combination of NMR relaxation and MD simulations can be used to create a microscopic picture of the solvent at the protein interface, which can be used for example in the study of chemical denaturation.

Place, publisher, year, edition, pages
Elsevier, 2010
Keyword
14N-urea NMR-relaxation, Molecular dynamics simulation, Urea–lysozyme system
Identifiers
urn:nbn:se:umu:diva-30184 (URN)10.1016/j.saa.2009.11.054 (DOI)000275584000002 ()20061179 (PubMedID)
Available from: 2009-12-10 Created: 2009-12-10 Last updated: 2010-04-20Bibliographically approved
3. The affect of urea on the kinetics of local unfolding processes in chymotrypsin inhibitor 2
Open this publication in new window or tab >>The affect of urea on the kinetics of local unfolding processes in chymotrypsin inhibitor 2
2010 (English)In: Biophysical Chemistry, ISSN 0301-4622, E-ISSN 1873-4200, Vol. 151, no 1-2, 46-53 p.Article in journal (Refereed) Published
Abstract [en]

The dynamics of chymotrypsin inhibitor 2 (CI2) in water, as well as in 10M urea, have been studied by Molecular Dynamics simulations. The analysis aims at investigating how local protein processes are affected by urea and how the perturbation by urea on the local level manifests itself in the kinetics of the global unfolding. The results show that the effect of urea on local processes depends upon the type of process at hand. An isolated two-residue contact on the surface of CI2 has a decreased frequency of rupture in the urea solvent. This is in contrast to the increased frequency of rupture of the hydrogen bonds in secondary structure elements in the urea solvent. It is proposed that the increase in the unfolding rates of complex protein processes is based upon the retardation of the refolding rate of small scale, isolated processes.

Keyword
urea, denaturation, chymotrypsin inhibitor 2, kinetics, molecular dynamics, MD simulation
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-35290 (URN)10.1016/j.bpc.2010.05.004 (DOI)000280510000007 ()20570033 (PubMedID)
Available from: 2010-08-11 Created: 2010-08-11 Last updated: 2010-10-04Bibliographically approved

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