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

Int J Mol Sci. 2021 Jan 5;22(1):445. doi: 10.3390/ijms22010445.


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.

Keywords: cell imaging; photochemistry; red fluorescent protein based Ca2+-biosensor; structure-activity relationships; ultrafast dynamics.

MeSH terms

  • Biosensing Techniques / methods
  • Calcium / metabolism*
  • Cell Line, Tumor
  • Fluorescence
  • Green Fluorescent Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Hydrogen Bonding
  • Luminescent Proteins / metabolism*
  • Protons
  • Spectrometry, Fluorescence / methods


  • Luminescent Proteins
  • Protons
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Calcium