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The Folding Energy Landscape of MerP
Umeå University, Faculty of Science and Technology, Department of Chemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4.

The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s-1, the folding rate was found to be unusually slow for a protein of this size.

The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H2O.

In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (< 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl.

Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.

Place, publisher, year, edition, pages
Umeå: Kemi , 2004. , 65 p.
Keyword [en]
Biochemistry, protein folding and stability, hydrogen exchange, intermediate, partial unfolding
Keyword [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-309ISBN: 91-7305-710-X (print)OAI: oai:DiVA.org:umu-309DiVA: diva2:143037
Public defence
2004-10-01, 10:00
Opponent
Supervisors
Available from: 2004-09-06 Created: 2004-09-06 Last updated: 2017-10-16Bibliographically approved
List of papers
1. Remarkably slow folding of a small protein
Open this publication in new window or tab >>Remarkably slow folding of a small protein
1997 In: FEBS Letters, Vol. 411, 359-364 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:umu:diva-4073 (URN)
Available from: 2004-09-06 Created: 2004-09-06Bibliographically approved
2. The “Two-State folder” MerP forms partially unfolded structures that show temperature dependent hydrogen exchange
Open this publication in new window or tab >>The “Two-State folder” MerP forms partially unfolded structures that show temperature dependent hydrogen exchange
Show others...
2004 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 340, no 2, 333-344 p.Article in journal (Refereed) Published
Abstract [en]

We have analysed the folding energy landscape of the 72 amino acid protein MerP by monitoring native state hydrogen exchange as a function of temperature in the range of 7-55 degrees C. The temperature dependence of the hydrogen exchange has allowed us to determine DeltaG, DeltaH and DeltaC(p) values for the conformational processes that permit hydrogen exchange. When studied with the traditional probes, fluorescence and CD, MerP appears to behave as a typical two-state protein, but the results from the hydrogen exchange analysis reveal a much more complex energy landscape. Analysis at the individual amino acid level show that exchange is allowed from an ensemble of partially unfolded structures (i.e. intermediates) in which the stabilities at the amino acid level form a broad distribution throughout the protein. The formation of partially unfolded structures might contribute to the unusually slow folding of MerP.

Place, publisher, year, edition, pages
London: Academic Press, 2004
Keyword
protein folding, hydrogen exchange, protein stability, intermediate, partially unfolded
Identifiers
urn:nbn:se:umu:diva-4074 (URN)10.1016/j.jmb.2004.05.003 (DOI)15201056 (PubMedID)
Available from: 2004-09-06 Created: 2004-09-06 Last updated: 2011-03-28Bibliographically approved
3. Hydrogen exchange in MerP reveals structure-dependent differences in compactness between the denatured states in GuHCl and H2O
Open this publication in new window or tab >>Hydrogen exchange in MerP reveals structure-dependent differences in compactness between the denatured states in GuHCl and H2O
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-4075 (URN)
Available from: 2004-09-06 Created: 2004-09-06 Last updated: 2012-05-04Bibliographically approved
4. The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data
Open this publication in new window or tab >>The equilibrium unfolding of MerP characterized by multivariate analysis of 2D NMR data
2005 (English)In: Journal of magnetic resonance, ISSN 1090-7807, E-ISSN 1096-0856, Vol. 172, no 1, 24-30 p.Article in journal (Refereed) Published
Abstract [en]

A general problem when analysing NMR spectra that reflect variations in the environment of target molecules is that different resonances are affected to various extents. Often a few resonances that display the largest frequency changes are selected as probes to reflect the examined variation, especially in the case, where the NMR spectra contain numerous resonances. Such a selection is dependent on more or less intuitive judgements and relying on the observed spectral variation being primarily caused by changes in the NMR sample. Second, recording changes observed for a few (albeit significant) resonances is inevitably accompanied by not using all available information in the analysis. Likewise, the commonly used chemical shift mapping (CSM) [Biochemistry 39 (2000) 26, Biochemistry 39 (2000) 12595] constitutes a loss of information since the total variation in the data is not retained in the projection into this single variable. Here, we describe a method for subjecting 2D NMR time-domain data to multivariate analysis and illustrate it with an analysis of multiple NNIR experiments recorded at various folding conditions for the protein MerP. The calculated principal components provide an unbiased model of variations in the NNIR spectra and they can consequently be processed as NMR data, and all the changes as reflected in the principal components are thereby made available for visual inspection in one single NMR spectrum. This approach is much less laborious than consideration of large numbers of individual spectra, and it greatly increases the interpretative power of the analysis.

Place, publisher, year, edition, pages
San Diego: Academic Press, 2005
Keyword
multivariate NMR data analysis, protein folding, PCA, PLS, GuHCl
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-13508 (URN)10.1016/j.jmr.2004.09.010 (DOI)000226176800004 ()
Available from: 2007-05-25 Created: 2007-05-25 Last updated: 2017-10-16Bibliographically approved

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