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 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.
Alvarez
Luis
L
Laboratoire de Chimie Physique, Université Paris-Sud, Orsay, France.
Levin
Chantal Houée
CH
Merola
Fabienne
F
Bizouarn
Tania
T
Pasquier
Hélène
H
Baciou
Laura
L
Rusconi
Filippo
F
Erard
Marie
M
D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D018384:Oxidative Stress
C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins