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Article

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

1
Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331-4003, USA
2
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada
3
Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(1), 445; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010445
Received: 8 December 2020 / Revised: 30 December 2020 / Accepted: 31 December 2020 / Published: 5 January 2021
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 (ΔR/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. View Full-Text
Keywords: red fluorescent protein based Ca2+-biosensor; photochemistry; ultrafast dynamics; structure-activity relationships; cell imaging red fluorescent protein based Ca2+-biosensor; photochemistry; ultrafast dynamics; structure-activity relationships; cell imaging
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MDPI and ACS Style

Tang, L.; Zhang, S.; Zhao, Y.; Rozanov, N.D.; Zhu, L.; Wu, J.; Campbell, R.E.; Fang, C. Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor. Int. J. Mol. Sci. 2021, 22, 445. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010445

AMA Style

Tang L, Zhang S, Zhao Y, Rozanov ND, Zhu L, Wu J, Campbell RE, Fang C. Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor. International Journal of Molecular Sciences. 2021; 22(1):445. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010445

Chicago/Turabian Style

Tang, Longteng, Shuce Zhang, Yufeng Zhao, Nikita D. Rozanov, Liangdong Zhu, Jiahui Wu, Robert E. Campbell, and Chong Fang. 2021. "Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor" International Journal of Molecular Sciences 22, no. 1: 445. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22010445

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