Green Fluorescent Protein Photobleaching: a Model for Protein Damage by Endogenous and Exogenous Singlet Oxygen

Abstract

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.

TransgenicLeishmaniaModel for Delta-Aminolevulinate-Inducible Monospecific Uroporphyria: Cytolytic Phototoxicity Initiated by Singlet Oxygen-Mediated Inactivation of Proteins and Its Ablation by Endosomal Mobilization of Cytosolic Uroporphyrin

“…8). A major role of 1 O 2 in this is indicated by the inhibition of GFP fluorescence bleaching by azide (22). Crucial residues for fluorescence of GFP (9,31) and catalytic function of NEO (19) may be modified by this ROS in the uroporphyric cells, accounting for their loss of these activities, as reported for several functional proteins in solution (11,18).…”

A general approach for chemical labeling and rapid, spatially controlled protein inactivation

“…5 D and G). Because photobleaching and ROS generation are parallel biophysical processes, this rapid photobleaching indicates that ROS are generated very quickly in this setting (25). This 15-s irradiation time was sufficient to inactivate FKBP12(F36V)-␤-gal (Fig.…”

Cryo‐fluorescence microscopy facilitates correlations between light and cryo‐electron microscopy and reduces the rate of photobleaching

“…It has been shown that excited fluorophores interact with dissolved oxygen producing reactive oxygen species, such as super oxide radicals (Wright et al, 2002). These can oxidize and destroy a variety of components, such as proteins and their associated fluorophores, leading to a loss of fluorescence signal (Greenbaum et al, 2000;Bernas et al, 2004). For this to happen, oxygen must collide with the excited dye.…”