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

Longteng Tang,Jiahui Wu,Yufeng Zhao,Robert E Campbell,Chong Fang,Nikita D Rozanov,Liangdong Zhu,Shuce Zhang

doi:10.3390/ijms22010445

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.

Highlights

Tracking intracellular Ca2+ is of paramount importance to cell biology since Ca2+ is a ubiquitous second messenger that regulates numerous cellular processes [1]
Several GCaMP-type single fluorescent protein (FP)-based ratiometric Ca2+ indicators have been engineered with improved dynamic ranges such as the monomeric GEX-GECO1, GEM-GECO1, and Y-GECOs [8,9]
For the total of 70 cells examined and pooled from three replications, an average ratio change of ~300% (∆R/R0, where R0 is the initial ratio) was achieved after background subtraction, while the signalto-noise ratio (SNR) in the green channel is above 3 with the mean filtering (Figure S10 in the SM). These imaging data substantiate the functionality of REX-GECO1 as a single FP-based emission-ratiometric Ca2+ indicator to be the second-generation genetically encoded calcium ion indicators (GECIs) with redder emission wavelengths that could greatly benefit bioimaging applications [40]

Summary

Introduction

Tracking intracellular Ca2+ is of paramount importance to cell biology since Ca2+ is a ubiquitous second messenger that regulates numerous cellular processes [1]. For the total of 70 cells examined and pooled from three replications, an average ratio change of ~300% (∆R/R0, where R0 is the initial ratio) was achieved after background subtraction, while the signalto-noise ratio (SNR) in the green channel is above 3 with the mean filtering (Figure S10 in the SM) These imaging data substantiate the functionality of REX-GECO1 as a single FP-based emission-ratiometric Ca2+ indicator (with room for further improvement) to be the second-generation genetically encoded calcium ion indicators (GECIs) with redder emission wavelengths that could greatly benefit bioimaging applications [40]

Femtosecond Transient Absorption Spectroscopy

Quantum Mechanical Calculations

Molecular Dynamics Simulations

Findings

Conclusions