How fluorescent proteins blink
Fluorescent proteins from the GFP family are remarkable markers for cell imaging. Their weak photostability, however, constitutes their principal disadvantage. If one observes under the microscope a single fluorescent molecule (a fluorescent protein, or an organic dye for example), blinking can be immediately noticed: fluorescence is not constant over time, but alternates between bright and dark periods. In the case of GFP’s, the molecular and structural origin of blinking remains mysterious. Excited states reactions can generate a transient loss of fluorescence, such as intersystem crossing to the triplet state, chromophore protonation, or chromophore isomerization. Another possibility consists in photo-induced electron transfer, which results in the production of a radical species that is unstable and nonfluorescent. In this work, we have provided evidence for such a radical species, which was generated by x-rays from the ESRF. By combining crystallography, Raman spectroscopy, and absorption and fluorescence spectroscopy, we could show that the radical state is characterized by a severe distortion of the chromophore, which accounts for the loss of fluorescence. This is the first study showing a fluorescent protein in a transiently off state. This study could allow the development of more photostable variants. The work also highlights the importance of electron transfer reactions in fluorescent proteins.
Virgile Adam, Philippe Carpentier, Sebastien Violot, Mickaël Lelimousin, Claudine Darnault, G. Ulrich Nienhaus & Dominique Bourgeois, "Structural Basis of X-ray Induced Photobleaching in a Photoactivatable Green Fluorescent Protein”, J. Am. Chem. Soc., (2009), 131:18063–18065 DOI: 10.1021/ja907296v.