Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?

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 pr...

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Published in:Photochemistry and photobiology 2010-01, Vol.86 (1), p.55-61
Main Authors: Alvarez, Luis, Levin, Chantal Houée, Merola, Fabienne, Bizouarn, Tania, Pasquier, Hélène, Baciou, Laura, Rusconi, Filippo, Erard, Marie
Format: Article
Language:English
Subjects:
Apoptosis
Aqueous solutions
Chemical Sciences
Fluorescence
Fluorescence Resonance Energy Transfer
Green Fluorescent Proteins - chemistry
Mass spectrometry
Nitric oxide
Oxidation
Oxidation-Reduction
Oxidative Stress
Physics
Proteins
Signal transduction
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creator Alvarez, Luis
Levin, Chantal Houée
Merola, Fabienne
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Rusconi, Filippo
Erard, Marie
description 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 W57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W57 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.
doi_str_mv 10.1111/j.1751-1097.2009.00617.x
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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 W57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W57 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. 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subjects Apoptosis
Aqueous solutions
Chemical Sciences
Fluorescence
Fluorescence Resonance Energy Transfer
Green Fluorescent Proteins - chemistry
Mass spectrometry
Nitric oxide
Oxidation
Oxidation-Reduction
Oxidative Stress
Physics
Proteins
Signal transduction
title Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress?
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