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 Wachter

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Abstract

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
JournalBiochemistry
Volume46
Issue number35
DOIs
StatePublished - Sep 4 2007

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Optical Imaging
Staphylococcal Protein A
Chromophores
Green Fluorescent Proteins
Tryptophan
Fluorescence
X-Rays
Imaging techniques
X rays
Conformations
Fluorescence Resonance Energy Transfer
Aspartic Acid
Fluorophores
Microscopy
Hydrogen
Nitrogen
Isomerization
Excited states
Ground state
Hydrogen bonds

ASJC Scopus subject areas

  • Biochemistry

Cite this

Malo, G. D., Pouwels, L. J., Wang, M., Weichsel, A., Montfort, W. R., Rizzo, M. A., ... Wachter, R. (2007). X-ray structure of Cerulean GFP: A tryptophan-based chromophore useful for fluorescence lifetime imaging. Biochemistry, 46(35), 9865-9873. https://doi.org/10.1021/bi602664c

X-ray structure of Cerulean GFP : A tryptophan-based chromophore useful for fluorescence lifetime imaging. / Malo, Gabrielle D.; Pouwels, Lauren J.; Wang, Meitian; Weichsel, Andrzej; Montfort, William R.; Rizzo, Mark A.; Piston, David W.; Wachter, Rebekka.

In: Biochemistry, Vol. 46, No. 35, 04.09.2007, p. 9865-9873.

Research output: Contribution to journalArticle

Malo, GD, Pouwels, LJ, Wang, M, Weichsel, A, Montfort, WR, Rizzo, MA, Piston, DW & Wachter, R 2007, 'X-ray structure of Cerulean GFP: A tryptophan-based chromophore useful for fluorescence lifetime imaging', Biochemistry, vol. 46, no. 35, pp. 9865-9873. https://doi.org/10.1021/bi602664c
Malo GD, Pouwels LJ, Wang M, Weichsel A, Montfort WR, Rizzo MA et al. X-ray structure of Cerulean GFP: A tryptophan-based chromophore useful for fluorescence lifetime imaging. Biochemistry. 2007 Sep 4;46(35):9865-9873. https://doi.org/10.1021/bi602664c
Malo, Gabrielle D. ; Pouwels, Lauren J. ; Wang, Meitian ; Weichsel, Andrzej ; Montfort, William R. ; Rizzo, Mark A. ; Piston, David W. ; Wachter, Rebekka. / X-ray structure of Cerulean GFP : A tryptophan-based chromophore useful for fluorescence lifetime imaging. In: Biochemistry. 2007 ; Vol. 46, No. 35. pp. 9865-9873.
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