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

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. Moerner, Partho Ghosh, Hiroshi Matsuo, Roger Y. Tsien, John Y. Lin and Erik A. Rodriguez ()
Additional contact information
Atanu Maiti: Frederick National Laboratory for Cancer Research
Cosmo Z. Buffalo: University of California, Berkeley
Saumya Saurabh: Stanford 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
John Y. Lin: University of Tasmania
Erik A. Rodriguez: The George Washington University

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-39776-9

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