Expanding the applications of dimerization-dependent fluorescent proteins

  • Author / Creator
    Lai, Yan Tiffany
  • The expansion of the fluorescent protein (FP) colour palette through discovery of more species possessing FPs and by subjecting them to intensive protein engineering efforts has greatly aided the effectiveness of FP-based biosensor designs. Currently, the main FP-based methods for detecting protein-protein interactions, enzymatic activity, and changes in small molecule concentration in live cells, are Förster resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC). A new biosensor strategy has recently been introduced by our group: dimerization-dependent FPs (ddFPs). This novel platform is based on the change in fluorescence intensity resulting from the reversible heterodimeric association of two distinct ddFP monomers. We believe that ddFPs can overcome some challenges in live cell imaging currently imposed by traditional biosensor platforms. For example, ddFPs may facilitate multicolour imaging of biosensors in live cells due to the fact that they have a narrower spectral profile than a FRET pair. In this thesis, we describe our efforts to expand the range of applications of ddFP in live cells. We report the creation of systems for the sensing of global O-linked β-N-acetylglucosamine (O-GlcNAc) modifications in live cells by O-GlcNAc transferase (OGT) and validation of OGT protease activity to host-cell factor-1 which may be the link between nutrient levels and the cell cycle. In addition, we apply ddFPs for sensing of tomosyn-1 SUMOylation by small ubiquitin modifier 1 (SUMO-1) in pancreatic islet cells. The creation of these plasmids is a proof of the versatility and modularity of the ddFP and its related FP exchange (FPX) biosensor design strategy. We also report the engineering of a dark “A” copy (DA) for ddFP applications. By a simple rational mutation of the chromophore of dimerization-dependent green A copy (GA), DA was generated. We confirmed that DA competes with GA for binding to the B copy. Specifically, we demonstrated the utility of DA in combination with GA and B as an intensiometric “turn on” biosensor for monitoring caspase-3 activity. Using both ddFPs and DA we endeavour to develop a facile multiparameter imaging platform.

  • Subjects / Keywords
  • Graduation date
    Fall 2015
  • Type of Item
  • Degree
    Master of Science
  • 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.