title: Are the fluorescent properties of the cyan fluorescent protein sensitive to conditions of oxidative stress?
abstract: The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative gamma-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.
authors.affiliation: Laboratoire de Chimie Physique, Université Paris-Sud, Orsay, France.
authors.forename: Luis
authors.initials: L
authors.lastname: Alvarez
authors.forename: Chantal Houée
authors.initials: CH
authors.lastname: Levin
authors.forename: Fabienne
authors.initials: F
authors.lastname: Merola
authors.forename: Tania
authors.initials: T
authors.lastname: Bizouarn
authors.forename: Hélène
authors.initials: H
authors.lastname: Pasquier
authors.forename: Laura
authors.initials: L
authors.lastname: Baciou
authors.forename: Filippo
authors.initials: F
authors.lastname: Rusconi
authors.forename: Marie
authors.initials: M
authors.lastname: Erard
mesh_terms: D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress
chemical_list: C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins