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.

To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins, we have determined the 2.1 Å X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan color class of Anthozoa fluorescent proteins, and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437-400 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the proteinintrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stablility. The refined crystal structure of the biological tetramer (spacegroup C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical to that in green fluorescent protein (avGFP). To test the roles of specific residues in color modulation, the optical properties of the H163Q and K70M variants were investigated. Although absorbance bands remain broad, peak excitation maxima are red-shifted to 455 and 460 nm, emitting cyan and green light respectively. To probe chromophore ground state features, Raman spectra were collected using 752 nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral avGFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan color is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.