X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging.
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.
Malo
Gabrielle D
GD
Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, USA.
Pouwels
Lauren J
LJ
Wang
Meitian
M
Weichsel
Andrzej
A
Montfort
William R
WR
Rizzo
Mark A
MA
Piston
David W
DW
Wachter
Rebekka M
RM
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000737:chemistry; D007202:Indicators and Reagents; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation / Q000379:methods*; D008958:Models, Molecular; D020283:Photoreceptors, Microbial; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins; D014364:Tryptophan / Q000737:chemistry*
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D007093:Imidazoles; D007202:Indicators and Reagents; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; C050162:photoactive yellow protein, Bacteria; D049452:Green Fluorescent Proteins; D006639:Histidine; D014364:Tryptophan