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Area of Science:

  • Biochemistry
  • Biophysics
  • Molecular Biology

Background:

  • Ratiometric indicators with long emission wavelengths are crucial for advanced bioimaging and life sciences.
  • Fluorescent protein (FP)-based biosensors offer advantages for in vivo imaging.
  • Understanding the working mechanism of novel biosensors is key to their optimization.

Purpose of the Study:

  • To elucidate the working mechanism of the red fluorescent protein (FP)-based calcium ion (Ca2+) biosensor, REX-GECO1.
  • To investigate the role of excited-state proton transfer in REX-GECO1's functionality.
  • To demonstrate the application of REX-GECO1 for Ca2+ imaging in human cells.

Main Methods:

  • Spectroscopic methods (e.g., fluorescence spectroscopy)
  • Computational methods
  • 2D-fluorescence mapping
  • Cell-based imaging assays

Main Results:

  • The Ca2+-free REX-GECO1 chromophore enters an excited dark state upon photoexcitation.
  • Ca2+ binding triggers ultrafast excited-state proton transfer via a hydrogen bond to Glu80, enabling functionality.
  • REX-GECO1 achieved Ca2+ imaging in HeLa cells with a ~300% red/green emission ratio change, outperforming existing indicators.

Conclusions:

  • The working mechanism of REX-GECO1 involves Ca2+-dependent excited-state proton transfer.
  • REX-GECO1 is a highly effective red fluorescent protein-based Ca2+ biosensor for bioimaging.
  • These findings facilitate the design of next-generation biosensors with enhanced dynamic range and longer emission wavelengths.