Singlet oxygen photosensitization by EGFP and its chromophore HBDI

The photosensitization of reactive oxygen species and, in particular, singlet oxygen by proteins from the green fluorescent protein (GFP) family influences important processes such as photobleaching and genetically targeted chromophore-assisted light inactivation. In this article, we report an investigation of singlet oxygen photoproduction by GFPs using time-resolved detection of the NIR phosphorescence of singlet oxygen at 1275 nm. We have detected singlet oxygen generated by enhanced (E)GFP, and measured a lifetime of 4 μs in deuterated solution. By comparison with the model compound of the EGFP fluorophore 4-hydroxybenzylidene-1,2-dimethylimidazoline (HBDI), our results confirm that the β-can of EGFP provides shielding of the fluorophore and reduces the production of this reactive oxygen species. In addition, our results yield new information about the triplet state of these proteins. The quantum yield for singlet oxygen photosensitization by the model chromophore HBDI is 0.004.