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Expanding the optogenetic toolkit with a photocleavable protein and a near infrared pH indicator

  • Author / Creator
    Zhang, Wei
  • Optogenetics is an emerging biological technique that is causing a revolution in life science. It uses optical methods to control and visualize genetically targeted biological events in a relatively non-invasive and spatiotemporally precise manner. Optogenetics requires advanced optical instruments, but the most important requirement is genetically encodable molecular tools to serve as the interface between light and biology. Naturally occurring photoreceptor proteins are a great source of optogenetic tools, however, their range of utility is restricted. Most naturally occurring photoreceptor proteins have required some degree of protein engineering for improved optical and functional properties. Encouragingly, an expanded range of optogenetic tools with novel functions has been developed by coupling the natural photosensory mechanisms with certain proteins. Through such efforts, the optogenetic toolkit is rapidly growing, though it is still relatively limited. In this thesis, we describe our efforts to expand the scope of photosensory mechanisms by engineering a photocleavable protein (PhoCl) that splits into two fragments upon violet light (~400 nm) illumination. PhoCl is a circularly permuted version of a green-to-red photoconvertible fluorescent protein (FP) in which the main chain break that accompanies photoconversion produces a short chromophore-containing peptide fragment that spontaneously dissociates from the large fragment. We have used PhoCl to manipulate protein localization and gene expression by genetically inserting it between a protein-of-interest (POI) and a subcellular localization tag. Upon illumination, the POI is released from the subcellular localization tag and is free to diffuse throughout the cell. We have also used PhoCl to “cage” an enzyme of interest by fusing a peptide-based inhibitor to an enzyme via PhoCl. Light-dependent cleavage of PhoCl leads to release of the inhibitor and activation of the formerly caged enzyme. This strategy has been successfully applied to hepatitis C virus (HCV) protease, which was further used to activate the channel-forming glycoprotein, Pannexin-1. We also report the development, optimization, and characterization of the first genetically encoded near infrared (NIR) fluorescent pH indicator, pH-mIFP. The pH dependent fluorescence was introduced to the NIR FP by rational design, which was followed by directed evolution for improved brightness and pH sensitivity. We have demonstrated pH-mIFP can report pH changes on the surface of cultured mammalian cells. Fluorescence imaging with long-wavelength excitation light has the advantages of low cytotoxicity, deeper tissues penetration, and spectral compatibility with existing optogenetic tools. Therefore, we expect pH-mIFP will find use for multicolour and deep-tissue fluorescence imaging of physiologically-relevant pH changes.

  • Subjects / Keywords
  • Graduation date
    2017-11:Fall 2017
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3Q23RD4M
  • 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
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Campbell, Robert (Department of Chemistry)
  • Examining committee members and their departments
    • Derda, Ratmir (Department of Chemistry)
    • Tucker, Chandra (Department of Pharmacology at University of Colorado School of Medicine)
    • McDermott, Mark (Department of Chemistry)
    • Cairo, Christopher (Department of Chemistry)