Development of cpRFP's for use as Ca2+ biosensors

  • Author / Creator
    Carlson, Haley J
  • 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.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • Le, Chris (Laboratory Medicine and Pathology)
    • Lutz, Stefan (Chemistry)
    • Lowary, Todd (Chemistry)
    • Lucy, Charles (Chemistry)