The structure of the chromophore within DsRed, a red fluorescent protein from coral

Proc Natl Acad Sci U S A. 2000 Oct 24;97(22):11990-5. doi: 10.1073/pnas.97.22.11990.


DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming C==N==C==O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C==N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C==N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.

Publication types

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

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid
  • Chromogenic Compounds / chemistry*
  • Cnidaria / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Luminescent Proteins / chemistry*
  • Mass Spectrometry
  • Protein Conformation
  • Protein Denaturation
  • Red Fluorescent Protein


  • Chromogenic Compounds
  • Luminescent Proteins