X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging

Biochemistry. 2007 Sep 4;46(35):9865-73. doi: 10.1021/bi602664c. Epub 2007 Aug 8.


The crystal structure of the cyan-fluorescent Cerulean green fluorescent protein (GFP), a variant of enhanced cyan fluorescent protein (ECFP), has been determined to 2.0 A. Cerulean bears an internal fluorophore composed of an indole moiety derived from Y66W, conjugated to the GFP-like imidazolinone ring via a methylene bridge. Cerulean undergoes highly efficient fluorescence resonance energy transfer (FRET) to yellow acceptor molecules and exhibits significantly reduced excited-state heterogeneity. This feature was rationally engineered in ECFP by substituting His148 with an aspartic acid [Rizzo et al. (2004) Nat. Biotechnol. 22, 445], rendering Cerulean useful for fluorescence lifetime imaging microscopy (FLIM). The X-ray structure is consistent with a single conformation of the chromophore and surrounding residues and may therefore provide a structural rationale for the previously described monoexponential fluorescence decay. Unexpectedly, the carboxyl group of H148D is found in a buried position, directly contacting the indole nitrogen of the chromophore via a bifurcated hydrogen bond. Compared to the similarly constructed ECFP chromophore, the indole group of Cerulean is rotated around the methylene bridge to adopt a cis-coplanar conformation with respect to the imidazolinone ring, resulting in a close edge-to-edge contact of the two ring systems. The double-humped absorbance spectrum persists in single-crystal absorbance measurements, casting doubt on the idea that ground state conformational heterogeneity forms the basis of the two overlapping transitions. At low pH, a blue shift in absorbance of 10-15 nm suggests a pH-induced structural transition that proceeds with a time constant of 47 (+/-2) min and is reversible. Possible interpretations in terms of chromophore isomerization are presented.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Crystallization
  • Crystallography, X-Ray*
  • Directed Molecular Evolution / methods
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry*
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / metabolism
  • Histidine / chemistry
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Imidazoles / chemistry
  • Indicators and Reagents
  • Microscopy, Fluorescence, Multiphoton / instrumentation
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Models, Molecular
  • Photoreceptors, Microbial
  • Protein Conformation
  • Protein Engineering
  • Recombinant Proteins
  • Tryptophan / chemistry*


  • Bacterial Proteins
  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Imidazoles
  • Indicators and Reagents
  • Photoreceptors, Microbial
  • Recombinant Proteins
  • photoactive yellow protein, Bacteria
  • Green Fluorescent Proteins
  • Histidine
  • Tryptophan

Associated data

  • PDB/2Q57