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GroEL-induced topological dislocation of a substrate protein β-sheet core: a solution EPR spin–spin distance study
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
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2010 (English)In: Journal of chemical biology, ISSN 1864-6158, E-ISSN 1864-6166, Vol. 3, no 3, 127-39 p.Article in journal (Refereed) Published
Abstract [en]

The Hsp60-type chaperonin GroEL assists in the folding of the enzyme human carbonic anhydrase II (HCA II) and protects it from aggregation. This study was aimed to monitor conformational rearrangement of the substrate protein during the initial GroEL capture (in the absence of ATP) of the thermally unfolded HCA II molten-globule. Single- and double-cysteine mutants were specifically spin-labeled at a topological breakpoint in the β-sheet rich core of HCA II, where the dominating antiparallel β-sheet is broken and β-strands 6 and 7 are parallel. Electron paramagnetic resonance (EPR) was used to monitor the GroEL-induced structural changes in this region of HCA II during thermal denaturation. Both qualitative analysis of the EPR spectra and refined inter-residue distance calculations based on magnetic dipolar interaction show that the spin-labeled positions F147C and K213C are in proximity in the native state of HCA II at 20 °C (as close as ∼8 Å), and that this local structure is virtually intact in the thermally induced molten-globule state that binds to GroEL. In the absence of GroEL, the molten globule of HCA II irreversibly aggregates. In contrast, a substantial increase in spin–spin distance (up to >20 Å) was observed within minutes, upon interaction with GroEL (at 50 and 60 °C), which demonstrates a GroEL-induced conformational change in HCA II. The GroEL binding-induced disentanglement of the substrate protein core at the topological break-point is likely a key event for rearrangement of this potent aggregation initiation site, and hence, this conformational change averts HCA II misfolding.

Place, publisher, year, edition, pages
2010. Vol. 3, no 3, 127-39 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-100534DOI: 10.1007/s12154-010-0038-2PubMedID: 21479077OAI: oai:DiVA.org:liu-100534DiVA: diva2:662952
Note

At the time of the licentiate theses defence this article was submitted.

Available from: 2013-11-08 Created: 2013-11-08 Last updated: 2017-12-06
In thesis
1. Molecular probes as protein analysis tools in biotechnology
Open this publication in new window or tab >>Molecular probes as protein analysis tools in biotechnology
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Protein-labeling today is a work of art, in vivo studies of proteins or other molecules can easily be performed, and the movement of the labeled molecule can be followed in real time. Labeling in vitro gives enormous amount of data in labs all over the world on a daily basis, where protein-protein, protein-DNA or other interactions are studied. Folding and unfolding events can be monitored wi th labels reporting on local or global environmental changes in a protein. The use of labeling seems endless, but in this thesis I have chosen to focus on two labeling techniques: spin-labeling and fluorescence labeling. Applying these techniques on protein-protein and protein-DNA interactions has resulted in better understanding of protein folding and function.

Chaperonin function at elevated temperatures

The model protein HCA II (259 amino acids) mainly consisting of a large 10 stranded ß-sheet with a topological breakpoint between strands 6 and 7. Two residues, adjacent in the folded structure and located at each side of this breakpoint, were used in a site-directed-spin-labeling (SDSL) experiment. The aim was to elucidate what happens at the breakpoint when the protein interacts with the chaperonin GroEL at elevated temperatures. The chaperonin GroEL is a 60 kDa protein known to aid protein folding in the cell. By probing the model protein, HCA II, we have shown that this chaperone can stretch its substrate and release it for a new refolding opportunity.

MexR protein interaction with DNA

MexR is a 147 amino acid protein dimer involved in transcriptional repression of the multidrug efflux operon MexAB-OprM in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Malfunction in MexR results in multidrug resistant bacteria resistant to therapeutic strategies. Site-specific fluorescence-labeling of MexR has been investigated as a means to provide a new strategy for localising DNA binding and quantifyi ng DNA affinity. ANS fluorescence of the MexR protein in the absence and presence of DNA, together with a range of biophysical measurements, has provided a new view on how MexR could be regulated by small molecule binding, and thus sheds new light on its functionality in gene repression.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2008. 35 p.
Series
Linköping Studies in Science and Technology. Thesis, ISSN 0280-7971 ; 1354
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-42271 (URN)62255 (Local ID)978-91-7393-955-3 (ISBN)62255 (Archive number)62255 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2013-11-08

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Owenius, RikardJarl, AnngelicaJonsson, Bengt-HaraldCarlsson, UnoHammarström, Per

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