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

doi:10.1038/s41467-023-39776-9

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

Nat Commun. 2023 Jul 12;14(1):4155. doi: 10.1038/s41467-023-39776-9.

Authors

Atanu Maiti¹, Cosmo Z Buffalo², Saumya Saurabh^{3

4}, Felipe Montecinos-Franjola⁵, Justin S Hachey⁵, William J Conlon⁵, Geraldine N Tran⁶, Bakar Hassan⁷, Kylie J Walters⁷, Mikhail Drobizhev⁸, W E Moerner³, Partho Ghosh⁹, Hiroshi Matsuo¹, Roger Y Tsien^{9

10}, John Y Lin¹¹, Erik A Rodriguez¹²

Affiliations

¹ Cancer Innovation Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
² Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, 94720, USA.
³ Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.
⁴ Department of Chemistry, New York University, New York, NY, 10003, USA.
⁵ Department of Chemistry, The George Washington University, Washington, DC, 20052, USA.
⁶ Department of Radiology, Boston University, Boston, MA, 02118, USA.
⁷ Protein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, 21702, USA.
⁸ Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, 59717, USA.
⁹ Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, USA.
¹⁰ Howard Hughes Medical Institute, La Jolla, CA, 92093, USA.
¹¹ Tasmanian School of Medicine, University of Tasmania, Hobart, Tasmania, 7000, Australia.
¹² Department of Chemistry, The George Washington University, Washington, DC, 20052, USA. erik_rodriguez@gwu.edu.

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.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, N.I.H., Intramural

MeSH terms

Biliverdine*
Coloring Agents*
Engineering
Luminescent Proteins / genetics
Red Fluorescent Protein

Substances

Luminescent Proteins
Biliverdine
Coloring Agents

Abstract

Publication types

MeSH terms

Substances

Grants and funding