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CL6mN: Rationally Designed Optogenetic Photoswitches with Tunable Dissociation Dynamics
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    CL6mN: Rationally Designed Optogenetic Photoswitches with Tunable Dissociation Dynamics
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    • Abhirup Mukherjee
      Abhirup Mukherjee
      Department of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
    • Chaitanya Sudrik
      Chaitanya Sudrik
      Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
    • Yuge Hu
      Yuge Hu
      Department of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
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    • Manish Arha
      Manish Arha
      Centre for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, United States
      More by Manish Arha
    • Mark Stathos
      Mark Stathos
      Bioengineering Graduate Program, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
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    • Jieung Baek
      Jieung Baek
      Department of Bioengineering, University of California, Berkeley, Berkeley, California 94720, United States
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    • David V. Schaffer*
      David V. Schaffer
      Department of Bioengineering, University of California, Berkeley, Berkeley, California 94720, United States
      Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720, United States
      Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California 94720, United States
      *Tel (510) 643-5963. Fax: 510-642-4778. Email: [email protected]
    • Ravi S. Kane*
      Ravi S. Kane
      Department of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
      *Tel. (404) 385-4608. Email: [email protected]
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    Other Access OptionsSupporting Information (4)

    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2020, 9, 9, 2274–2281
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    https://doi.org/10.1021/acssynbio.0c00362
    Published August 14, 2020
    Copyright © 2020 American Chemical Society

    Abstract

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    The field of optogenetics uses genetically encoded photoswitches to modulate biological phenomena with high spatiotemporal resolution. We report a set of rationally designed optogenetic photoswitches that use the photolyase homology region of A. thaliana cryptochrome 2 (Cry2PHR) as a building block and exhibit highly efficient and tunable clustering in a blue-light dependent manner. CL6mN (Cry2-mCherry-LRP6c with N mutated PPPAP motifs) proteins were designed by mutating and/or truncating five crucial PPP(S/T)P motifs near the C-terminus of the optogenetic Wnt activator Cry2-mCherry-LRP6c, thus eliminating its Wnt activity. Light-induced CL6mN clusters have significantly greater dissociation half-lives than clusters of wild-type Cry2PHR. Moreover, the dissociation half-lives can be tuned by varying the number of PPPAP motifs, with the half-life increasing as much as 6-fold for a variant with five motifs (CL6m5) relative to Cry2PHR. Finally, we demonstrate the compatibility of CL6mN with previously reported Cry2-based photoswitches by optogenetically activating RhoA in mammalian cells.

    Copyright © 2020 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.0c00362.

    • Supplementary Figure S1: Characterization of the ability to activate Wnt signaling in CL6mN-transfected cells; Supplementary Figure S2: Characterization of the effect of CL6m5 on noncanonical Wnt pathways; Supplementary Figure S3: Comparison of fluorescence intensity for cells transfected with Cry2PHR and CL6mNs; Supplementary Figure S4: Characterization of number of clusters for cells transfected with Cry2PHR and CL6mNs normalized with respect to the cellular area; Supplementary Figure S5: Characterization of dissociation half-life for Cry2PHR-GFP (PDF)

    • Supplementary Video S1: A comparison of dissociation dynamics of Cry2PHR-mCherry, CL6m3, and CL6m5 at 25 °C in the RFP channel, time format in minute:second (scale bar, 10 μm) (AVI)

    • Supplementary Video S2: CL6m3-mediated clustering of CIBN-cyto (scale bar, 10 μm) (AVI)

    • Supplementary Video S3: CL6m5-mediated clustering of CIBN-cyto (scale bar, 10 μm) (AVI)

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    This article is cited by 3 publications.

    1. Pamela L. Toledo, Alejo R. Gianotti, Diego S. Vazquez, Mario R. Ermácora. Protein nanocondensates: the next frontier. Biophysical Reviews 2023, 15 (4) , 515-530. https://doi.org/10.1007/s12551-023-01105-1
    2. Amelia C. McCue, Brian Kuhlman. Design and engineering of light-sensitive protein switches. Current Opinion in Structural Biology 2022, 74 , 102377. https://doi.org/10.1016/j.sbi.2022.102377
    3. Diego S. Vazquez, Pamela L. Toledo, Alejo R. Gianotti, Mario R. Ermácora. Protein conformation and biomolecular condensates. Current Research in Structural Biology 2022, 4 , 285-307. https://doi.org/10.1016/j.crstbi.2022.09.004

    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2020, 9, 9, 2274–2281
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.0c00362
    Published August 14, 2020
    Copyright © 2020 American Chemical Society

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