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Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase

Nature Chemical Biology volume 12, pages 425430 (2016) | Download Citation

Abstract

Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein–protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2–CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2–CIB system by identifying versions of CRY2–CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13–α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

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Acknowledgements

We thank Dr. Constance Cepko for the pCALVL-dsRed Cre reporter (13769), obtained through Addgene, Dr. Matthew Kennedy for critical reading of the manuscript, and Jessica Spiltoir and Qi Liu for experimental assistance. This work was supported by grants from the National Institutes of Health (GM100225) and the McKnight Endowment Fund for Neuroscience (Technological Innovations in Neuroscience Award) to C.L.T.

Author information

Affiliations

  1. Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA.

    • Amir Taslimi
    • , Jose G Miranda
    • , Gopal P Pathak
    •  & Chandra L Tucker
  2. Department of Chemistry, Southern Methodist University, Dallas, Texas, USA.

    • Brian Zoltowski
  3. Department of Biology, Duke University, Durham, North Carolina, USA.

    • Robert M Hughes

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Contributions

A.T., B.Z., J.G.M., G.P.P., R.M.H., and C.L.T. carried out experiments. B.Z. carried out protein structural characterization and in vitro studies with OtCPF1. A.T., B.Z., and C.L.T. analyzed data and wrote the manuscript. C.L.T. conceived the project and edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Chandra L Tucker.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Results, Supplementary Figures 1–7.

Excel files

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    Supplementary Table 1

    Sequences of constructs used in studies

Videos

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    Recruitment and dissociation of CRY2PHR-mCh with CIB81-pmEGFP

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DOI

https://doi.org/10.1038/nchembio.2063

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