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2q57

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{{STRUCTURE_2q57| PDB=2q57 | SCENE= }}
{{STRUCTURE_2q57| PDB=2q57 | SCENE= }}
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'''X-ray structure of Cerulean GFP: A tryptophan-based chromophore useful for fluorescence lifetime imaging'''
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===X-ray structure of Cerulean GFP: A tryptophan-based chromophore useful for fluorescence lifetime imaging===
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==Overview==
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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.
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{{ABSTRACT_PUBMED_17685554}}
==About this Structure==
==About this Structure==
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[[Category: Beta barrel]]
[[Category: Beta barrel]]
[[Category: Fluorescent protein]]
[[Category: Fluorescent protein]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 20:01:02 2008''

Revision as of 17:01, 28 July 2008

Image:2q57.png

Template:STRUCTURE 2q57

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

Template:ABSTRACT PUBMED 17685554

About this Structure

2Q57 is a Single protein structure of sequence from Aequorea victoria. Full crystallographic information is available from OCA.

Reference

X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging., Malo GD, Pouwels LJ, Wang M, Weichsel A, Montfort WR, Rizzo MA, Piston DW, Wachter RM, Biochemistry. 2007 Sep 4;46(35):9865-73. Epub 2007 Aug 8. PMID:17685554

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