Development of fluorogenic fluorescent protein heterodimers

  • Author / Creator
    Alford, Spencer Caleb
  • Fluorescent proteins (FPs) are indispensible biochemical tools. The concerted efforts of protein engineers have produced FPs spanning the visible colour spectrum. This wide variety of FPs has greatly facilitated the development of FP-based biosensors. However, researchers rely on relatively few fundamental biosensor design templates. Förster resonance energy transfer and bimolecular complementation are the principal FP-based technologies for live cell imaging of physiological events, such as changes in small molecule concentration, enzymatic activities, and protein-protein interactions. Although widely used, these techniques are often restrictive due to poor signal-to-noise ratios and irreversible sensing, respectively. Furthermore, examples of these biosensor strategies incorporating red FPs are limited. In this thesis we describe our efforts to address this shortcoming in the area of FP-based biosensors. We developed a dimerization-dependent red FP (ddRFP) that serves as an alternative template for biosensor construction. The prototype ddRFP was engineered from a homodimeric variant of a Discosoma red FP. Through extensive directed evolution the homodimer was converted into a fluorogenic obligate heterodimer. The reversible changes in fluorescence intensity that result from association of the ddRFP monomeric constituents, or the irreversible decrease that accompanies dissociation of covalently linked partners following linker cleavage, provides a useful spectroscopic signal for biosensing applications. Specifically, we demonstrated that ddRFP is useful for detecting in vitro protein-protein interactions, as well as imaging changes in calcium ion concentration and activation of caspase-3 in live cells. We also report the expansion of the ddFP colour palette through the development of green (ddGFP) and yellow (ddYFP) ddFP variants. These variants have several improvements relative to the ddRFP prototype including increased in vitro contrast and brightness for ddGFP, and a reduced pKa for ddYFP. While their utility for some live cell imaging applications is restricted due to low dissociation constants, ddGFP proved to be a useful fluorescent label of intermembrane contact sites between the endoplasmic reticulum and the mitochondrial network.

  • 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
    • Dr. J. Harrison (Chemistry)
    • Dr. J. Gibbs-Davis (Chemistry)
    • Dr. M. Glover (Biochemistry)
    • Dr. J. Pelletier (Chemistry) ; external (University of Montreal)
    • Dr. T. Lowary (Chemistry)