Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca 2+ Biosensor

• Published in: International journal of molecular sciences Vol. 22; no. 1
• Main Authors: Tang, Longteng, Zhang, Shuce, Zhao, Yufeng, Rozanov, Nikita D, Zhu, Liangdong, Wu, Jiahui, Campbell, Robert E, Fang, Chong
• Format: Journal Article
• Language: English
• Published: Switzerland 05.01.2021
• Subjects: Biosensing Techniques - methods; Calcium - metabolism; Cell Line, Tumor; Fluorescence; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Hydrogen Bonding; Luminescent Proteins - metabolism; Protons; Red Fluorescent Protein; Spectrometry, Fluorescence - methods; red fluorescent protein based Ca2+-biosensor; structure-activity relationships; photochemistry; ultrafast dynamics; cell imaging

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.