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Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor.


ABSTRACT: 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 Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ 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 Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (?R/R0) 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.

SUBMITTER: Tang L 

PROVIDER: S-EPMC7794744 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca<sup>2+</sup> Biosensor.

Tang Longteng L   Zhang Shuce S   Zhao Yufeng Y   Rozanov Nikita D ND   Zhu Liangdong L   Wu Jiahui J   Campbell Robert E RE   Fang Chong C  

International journal of molecular sciences 20210105 1


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<sup>2+</sup> biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca<sup>2+</sup>-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca<sup>2+</sup> switches the route to ultrafast excited-state  ...[more]

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