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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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    Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, and Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennesee 37232
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    Biochemistry

    Cite this: Biochemistry 2007, 46, 35, 9865–9873
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    https://doi.org/10.1021/bi602664c
    Published August 8, 2007
    Copyright © 2007 American Chemical Society
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    Abstract

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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 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.

    Copyright © 2007 American Chemical Society

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    †

     This work was supported by a grant from the National Science Foundation (NSF Grant MCB-0615938) to R.M.W., and a grant from the National Institutes of Health (NIH HL62969) to W.R.M.

    ‡

     Coordinates and structure factors for Cerulean GFP have been deposited in the Protein Data Bank (entry ID 2Q57).

    #

     Arizona State University.

    §

     Current address:  Swiss Light Source, Paul Scherrer Institut, Villigen PSI, Switzerland 5232.

    ⊥

     University of Arizona.

    ‖

     Current address:  Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201.

    ○

     Vanderbilt University.

    *

     Corresponding author. Phone:  480-965-8188; fax 480-965-2747; e-mail:  [email protected].

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    Biochemistry

    Cite this: Biochemistry 2007, 46, 35, 9865–9873
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi602664c
    Published August 8, 2007
    Copyright © 2007 American Chemical Society
    Request reuse permissions

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