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Theses and Dissertations

Development of cpRFP's for use as Ca2+ biosensors Open Access


Other title
fluorescent protein
Type of item
Degree grantor
University of Alberta
Author or creator
Carlson, Haley J
Supervisor and department
Campbell, Robert E (Chemistry)
Examining committee member and department
Lutz, Stefan (Chemistry)
Le, Chris (Laboratory Medicine and Pathology)
Lowary, Todd (Chemistry)
Lucy, Charles (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
The discovery of green fluorescent protein (GFP) from the Aequorea victoria jellyfish revolutionized many fields in the scientific community, including molecular biology, protein engineering, and neuroscience. The ability to genetically link a fluorescent protein to a protein of interest has allowed scientists to probe the exact structural localization of proteins. Another important application of FPs is their design for use in biosensors, whereby the fluorescence of the protein is intrinsically dependent on a small molecule of interest, such as calcium ion (Ca2+) or a physiological process such as phosphorylation or caspase activity. In single FP-based biosensors of small molecules, the FP must be circular permutated, whereby the original N- and C-termini are linked together and new termini are introduced closer to the chromophore. At the start of the work described in this thesis a lot of work had gone into developing and improving GFP-based Ca2+ biosensors1,2, but there were no reports of a red FP-based biosensor. The work in this thesis describes the engineering of an RFP-based Ca2+ biosensor using a circular permutated RFP, mCherry. The first step in this process was to engineer a cpmCherry variant with termini near the chromophore3. mCherry required a lot of engineering and optimization in order to identify a fluorescent variant with termini near the chromophore. Ultimately, a cpmCherry split at position 145 was found that, when fused to calmodulin (CaM) and M13, showed a response to Ca2+. The initial construct had limited response and was subjected to several rounds of mutagenesis to improve both the brightness and fluorescence response. The final variant CH-GECO3.1 shows a 250% signal increase with Ca2+ and could be imaged successfully in mammalian cells to monitor Ca2+ fluctuations. To further our understanding of this biosensor, site-directed mutagenesis was done to probe the structure-function relationship. After mutagenesis a few residues stood out as key residues that likely played a role in the mechanism of fluorescence increase, such as Gln163 and Glu61 (linker). Other mutations were introduced into the protein to determine whether the excitation and emission wavelengths could be altered, while still retaining function. The final section of this work describes the reconstitution of split green and red Ca2+ biosensors using intein technology. Inteins will spontaneously splice together protein fragments that are genetically linked to them. To take advantage of protein splicing several different Ca2+ biosensors were split into and N-terminal and C-terminal fragments and attached to the N-terminal or C-terminal intein, respectively. These fragments were co-transfected into mammalian HeLa cells and imaged for fluorescence signal and response to Ca2+ fluctuations.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Carlson, H.J. Campbell, R.E. 2011 Curr Opin Biotechnol: 20, 19-27.Carlson, H.J. Cotton, D.W. Campbell, R.E. 2010 Protein Sci: 19, 1490-1499.

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