Here, we show that irreversible bleaching of two different variants of GFP (sfGFP [FPbase: Superfolder GFP], EGFP [FBbase: sg25]) with visible light is paralleled by successive backbone fragmentation of the protein. Mass spectrometry revealed that the site of fragmentation resides at the fluorophore, between residue positions 65 and 66.
To summarize, irradiation-induced protein fragmentation can be achieved with blue, visible light for two common GFP variants. Presumably, photolysis can also occur at 488 nm, a wavelength common on standard microscopes, provided the intensity of illumination is sufficient. The site of fragmentation maps between position 65 and 66 of the fluorophore, where light absorption is translated to bond breakage.