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J. Biol. Chem., Vol. 277, Issue 52, 50573-50578, December 27, 2002
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From the Yellow emission variants of green fluorescent
protein (GFP) have been found useful in a variety of applications in
biological systems due to their red-shifted emission spectrum and
sensitivity to environmental parameters, such as pH and ionic strength.
However, slow maturation properties and new requirements for more
intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by
introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 Å resolution, which enabled us to correlate its novel features with these mutation points.
The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at
37 °C by removing steric and energetic constraints, which may hinder
folding of the polypeptide chain, and by accelerating the oxidation of
the C
Crystal Structure of Venus, a Yellow Fluorescent
Protein with Improved Maturation and Reduced Environmental
Sensitivity*
§,¶,
**,
Division of Molecular and Structural
Biology, Ontario Cancer Institute and Department of Medical Biophysics,
University of Toronto, Toronto, Ontario M5G 2M9, Canada,
Structure and Function of Biomolecules, PRESTO, JST, Nittochi
535 Akinono-cho Nakagyo-ku, Kyoto 604-0847, Japan, and the
** Laboratory for Cell Function and Dynamics, Advanced
Technology Development Center, Brain Science Institute, RIKEN,
Wako-city, Saitama 351-0198, Japan
-C
bond of Tyr66 during chromophore
formation. M153T, V163A, and S175G were also found to improve the rate
of maturation by creating regions of greater flexibility. F64L induced
large conformational changes in the molecule, leading to the removal of
halide sensitivity by preventing ion access to the binding site.
*
This work was supported in part by the grant from Howard
Hughes Medical Institute under the International Research Scholar program (to M. I.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Canadian Institutes of Health Research Investigator. To whom
correspondence should be addressed. Fax: 416-946-2055 (or 6529); E-mail: mikura@uhnres.utoronto.ca.
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