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

Author:

Maiti AtanuORCID,Buffalo Cosmo Z.,Saurabh Saumya,Montecinos-Franjola FelipeORCID,Hachey Justin S.,Conlon William J.,Tran Geraldine N.,Hassan Bakar,Walters Kylie J.ORCID,Drobizhev MikhailORCID,Moerner W. E.ORCID,Ghosh ParthoORCID,Matsuo HiroshiORCID,Tsien Roger Y.,Lin John Y.ORCID,Rodriguez Erik A.ORCID

Abstract

AbstractThe 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.

Funder

GW | Dr. Cyrus and Myrtle Katzen Cancer Research Center, George Washington University

American Heart Association

U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke

U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences

U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases

U.S. Department of Health & Human Services | NIH | National Cancer Institute

Publisher

Springer Science and Business Media LLC

Subject

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

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