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, Erik A. Rodriguez

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article number4155
JournalNature communications
Issue number1
StatePublished - Dec 2023

ASJC Scopus subject areas

  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology
  • General Physics and Astronomy


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