Site-Specific Fragmentation of Green Fluorescent Protein Induced by Blue Light

Green fluorescent protein (GFP) and related fluorescent proteins have multiple applications in cell biology, and elucidating their functions has been at the focus of biophysical research for about three decades. Fluorescent proteins can be bleached by intense irradiation, and a number of them undergo photoconversion. Rare cases have been reported where distant functional relatives of GFP exhibit UV-light-induced protein fragmentation. Here, we show that irreversible bleaching of two different variants of GFP (sfGFP, EGFP) 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.