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 (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.

Encoding, transmission, decoding, and specificity of calcium signals in plants

“…Ratiometric calcium indicators include Fura-2 acetoxymethyl ester, Fura-Red acetoxymethyl ester, Fura-2 pentapotassium, Indo-1 acetoxymethyl ester, and Indo-1 pentapotassium ( Tinning et al , 2018 ). These dyes exhibit a shift in emission or excitation spectrum upon calcium binding ( Tang et al , 2021 ). Utilizing ‘ratio’ technology yields a more accurate readout for quantitative real-time measurements, although such chemical treatments are not suited for long-term imaging.…”

Fluorescence Modulation by Ultrafast Chromophore Twisting Events: Developing a Powerful Toolset for Fluorescent-Protein-Based Imaging

“…The subsequent logical question concerns other FP-based biosensors, ideally with redder emission wavelengths, as desired by the bioimaging and bioengineering communities due to deeper penetration depths, diminishment of autofluorescence, and reduced photo- and cytotoxicity. ,, We thus shifted our attention to another recently developed red fluorescent protein (RFP)-based Ca 2+ biosensor, REX-GECO1, and observed more dramatic chromophore twisting events . REX-GECO1 is an intensiometric and excitation-ratiometric bioprobe with a large Stokes shift and dynamic range, benefiting the signal-to-noise ratio for super-resolution microscopy .…”

“…However, most of these studies were focused on anionic p -HBDI and/or its derivatives in vacuo or in solution, and debates remain regarding the exact light-induced pathways, such as sequential or parallel roles played by P-ring and imidazolinone ring (I-ring) twisting motions (see Figure , left panel), and whether or not these processes are barrierless on ultrafast time scales . In contrast, our recent works have targeted and directly observed the onset, progression, and outcome of the chromophore twisting events inside the protein matrix in a series of FP derivatives and FP-based biosensors, ,− as well as photoconvertible and photoswitchable proteins …”

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

“…To address the issue that red-shifted FPs require two photon laser excitation with wavelengths outside the near-infrared region (>1000 nm), the long Stokes shift Ca 2+ sensor REX-GECO1 has been engineered [73]. REX-GECO1 can be excited by one 480 nm photon or two 910 nm photons and has been demonstrated to act as both an excitation as well as an emission ratiometric single FP-based Ca 2+ probe [74].…”