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Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?
Author(s) -
Alvarez Luis,
Levin Chantal Houée,
Merola Fabienne,
Bizouarn Tania,
Pasquier Hélène,
Baciou Laura,
Rusconi Filippo,
Erard Marie
Publication year - 2010
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2009.00617.x
Subject(s) - cyan , fluorescence , oxidative stress , biophysics , yellow fluorescent protein , fluorescent protein , chemistry , green fluorescent protein , oxidative phosphorylation , förster resonance energy transfer , biochemistry , biology , optics , physics , gene
The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ‐radiolysis, we have studied the ROS‐induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP‐HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W 57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W 57 as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.