An improved cyan fluorescent protein variant useful for FRET

Nat Biotechnol. 2004 Apr;22(4):445-9. doi: 10.1038/nbt945. Epub 2004 Feb 29.


Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

Publication types

  • Letter
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / chemistry*
  • COS Cells
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Fluorescence Resonance Energy Transfer / methods*
  • Glucokinase / metabolism
  • Green Fluorescent Proteins
  • Hydrogen-Ion Concentration
  • Indicators and Reagents / pharmacology
  • Light
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / pharmacology
  • Microscopy, Fluorescence
  • Mutagenesis, Site-Directed
  • Mutation
  • Recombinant Proteins / chemistry
  • Spectrophotometry
  • Time Factors


  • Bacterial Proteins
  • Indicators and Reagents
  • Luminescent Proteins
  • Recombinant Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • Glucokinase