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TitleSwitching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca Biosensor.
Publication TypeJournal Article
Year of Publication2021
AuthorsTang, Longteng, Shuce Zhang, Yufeng Zhao, Nikita D. Rozanov, Liangdong Zhu, Jiahui Wu, Robert E. Campbell, and Chong Fang
JournalInt J Mol Sci
Date Published2021 Jan 05

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor's functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.

Alternate JournalInt J Mol Sci
PubMed ID33466257
PubMed Central IDPMC7794744
Grant ListMCB-1817949 / / National Science Foundation /
DMR-1920368 / / National Science Foundation /