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Structural and photophysical characterization of the small ultra-red fluorescent protein

Author

Listed:
  • Atanu Maiti

    (Frederick National Laboratory for Cancer Research)

  • Cosmo Z. Buffalo

    (University of California, Berkeley)

  • Saumya Saurabh

    (Stanford University
    New York University)

  • Felipe Montecinos-Franjola

    (The George Washington University)

  • Justin S. Hachey

    (The George Washington University)

  • William J. Conlon

    (The George Washington University)

  • Geraldine N. Tran

    (Boston University)

  • Bakar Hassan

    (National Institutes of Health)

  • Kylie J. Walters

    (National Institutes of Health)

  • Mikhail Drobizhev

    (Montana State University)

  • W. E. Moerner

    (Stanford University)

  • Partho Ghosh

    (University of California, San Diego)

  • Hiroshi Matsuo

    (Frederick National Laboratory for Cancer Research)

  • Roger Y. Tsien

    (University of California, San Diego
    Howard Hughes Medical Institute)

  • John Y. Lin

    (University of Tasmania)

  • Erik A. Rodriguez

    (The George Washington University)

Abstract

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

Suggested Citation

  • Atanu Maiti & Cosmo Z. Buffalo & Saumya Saurabh & Felipe Montecinos-Franjola & Justin S. Hachey & William J. Conlon & Geraldine N. Tran & Bakar Hassan & Kylie J. Walters & Mikhail Drobizhev & W. E. Mo, 2023. "Structural and photophysical characterization of the small ultra-red fluorescent protein," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-39776-9
    DOI: 10.1038/s41467-023-39776-9
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    References listed on IDEAS

    as
    1. Robert M. Dickson & Andrew B. Cubitt & Roger Y. Tsien & W. E. Moerner, 1997. "On/off blinking and switching behaviour of single molecules of green fluorescent protein," Nature, Nature, vol. 388(6640), pages 355-358, July.
    2. Ishan Deshpande & Jiahao Liang & Danielle Hedeen & Kelsey J. Roberts & Yunxiao Zhang & Betty Ha & Naomi R. Latorraca & Bryan Faust & Ron O. Dror & Philip A. Beachy & Benjamin R. Myers & Aashish Mangli, 2019. "Smoothened stimulation by membrane sterols drives Hedgehog pathway activity," Nature, Nature, vol. 571(7764), pages 284-288, July.
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