Abstract :

Fluorescent proteins (FPs) of the GFP family, combined with recent advances in genetic engineering and microscopy techniques, have revolutionized biological imaging. They are used as optical reporters in genetically encoded biosensors to visualize specific molecular activities in living cells. Unfortunately, the sensitivity of these biosensors remains limited by their low photostability under illumination. This is mainly due to the fact that the photochemical reactions responsible for FP photobleaching are still poorly characterized, making the rational optimization of their photostability difficult. This study clarifies the photobleaching mechanism of fluorescent proteins, particularly that of Citrine, a yellow FP widely used in cellular imaging. Thanks to the development of a dedicated instrumental setup, the photobleaching of several FPs could be quantitatively characterized using parameters such as the photobleaching quantum yield and the relative amounts of photoproducts exhibiting distinct spectroscopic signatures. Mass spectrometry analysis of photobleached FPs also revealed a large diversity of photoproducts. Detailed analysis of these products revealed, for Citrine, two main photobleaching mechanisms: the first is oxygen-dependent and involves oxidation of several residues and chromophore protonation. The second involves cleavage of the Citrine polypeptide chain near the chromophore, resulting in its destruction.