Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry

Abstract

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4{degrees}C and a few days at 37{degrees}C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.