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

Gabrielle D. Malo, Lauren J. Pouwels, Meitian Wang, Andrzej Weichsel, William R. Montfort, Mark A. Rizzo, David W. Piston, Rebekka M. Wachter

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


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 Å. 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.

Original languageEnglish (US)
Pages (from-to)9865-9873
Number of pages9
Issue number35
StatePublished - Sep 4 2007

ASJC Scopus subject areas

  • Biochemistry


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