Engineering of red fluorescent proteins and red fluorescent protein-based pH biosensors

  • Author / Creator
    Shen, Yi
  • Fluorescent proteins (FP) and FP-based biosensors have become essential tools for cell biology and neuroscience research. This thesis describes efforts to engineer new FPs with red emission and novel genetically encoded biosensors based on red FPs. Directed evolution and semi-rational design are the main techniques used to develop novel and improved red FP and red FP-based biosensors. First, A new pH-sensitive red fluorescent protein based on mApple, termed as “pHuji”, has been developed with high pH sensitivity, exhibiting over 20-fold fluorescence intensity change between pH 5.5 and pH 7.5. In live cell imaging, cell surface-displayed pHuji demonstrated high pH sensitivity when exposed to buffers with defined pH values. Collaborators have used pHuji for successful visualization of pH changes during exocytosis and endocytosis. Following the engineering of intensiometric red pH sensors, we next turned our attention to ratiometric red pH sensors. Through a process of semi-rational design and directed evolution, the red FP mApple was successfully engineered into a series of dual-excitation, ratiometric pH sensors. These new red-shifted ratiometric pH sensors, termed pHlorina, exhibit large ratio change (over 70-fold) for pH changes from 5.0 to 7.5. A series of long Stokes shift variants of mApple (λex = 450 nm and λem = 610 nm) were also developed and characterized. A photochromic and thermochromic red FP was serendipitously discovered during the process of engineering the long Stokes shift red FP. This protein, which we designated as switchable hypersensitive red FP (shyRFP), was characterized in terms of light, temperature, and pH dependence. A colour switching mechanism that involves protonation coupled E-Z isomerization of the protein chromophore was proposed. The monomeric RFP mCherry is widely used for live cell fluorescence imaging experiments. Using semi-rational design and random mutagenesis, two new mCherry variants were developed: Long Stokes Shift mCherry (LSSmCherry; λex = 460 nm and λem = 610 nm) and Red-Shifted mCherry (RDSmCherry; λex = 600 nm and λem = 630 nm). These two proteins have distinctively different fluorescence excitation and emission profiles from their predecessor mCherry2. These new additions to the FP toolbox provide templates for the engineering of new colour variants and fluorescent sensors with red emission. Finally, a filter paper-based screening method has been developed to screen for calcium ion (Ca2+) sensitive RFP variants expressed in Escherichia coli colonies. These low-affinity Ca2+ sensors were semi-rationally designed by altering protein barrel residues near the chromophore to form a Ca2+ binding pocket. By combining high-throughput screening and rational design, a number of Ca2+ sensitive variants were successfully identified.

  • Subjects / Keywords
  • Graduation date
    Spring 2015
  • 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
    • Harynuk, James (Chemistry)
    • Chica, Roberto (Chemistry, University of Ottawa)
    • Klassen, John (Chemistry)
    • Serpe, Michael (Chemistry)