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AEDANS labeled MexR variants and their DNA interaction
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. 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, Molecular Biotechnology. 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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(English)Manuscript (preprint) (Other academic)
Abstract [en]

MexR is a transcriptional repressor of the multidrug efflux-pump operon in the bacterial pathogen Pseudomonas aeruginosa, and thus, malfunction in the DNA-binding of the MexR protein leads to multidrug resistance (Poole et al., 1996). lt has been shown that mutations in the MexR protein lead to lost DNA binding ability (Saito et al., 2003). X-ray studies of the MexR protein (Lim et al., 2002) show that the protein contains a winged helix-turn-helix motif and this type of motif is known to be involved in DNA-binding (Gajiwala & Burley, 2000), but no exact DNA-binding sequence in MexR has yet been determined.

This study attempts to identify regions in the MexR protein involved in DNA-binding using a molecular probe as mapping tool. Three cysteines naturally occur in the MexR protein, located in positions 30, 62 and 138, respectively, and we have used two of them to map the DNA-binding site. Preliminary results show that the fluorescence of an TAEDANS probe attached to cys 62 is affected by DNA-binding, while a probe at cys 30 is not affected. Further studies are required to analyse the implications of these results.

National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-100536OAI: oai:DiVA.org:liu-100536DiVA: diva2:662979
Note

Progress report on MexRwt DNA binding studies

Available from: 2013-11-08 Created: 2013-11-08 Last updated: 2013-11-08
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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Jarl, AnngelicaAndrésen, CeciliaSvensson, MagdalenaSunnerhagen, Maria

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