Structural basis of X-ray induced transient photobleaching in a photoactivatable green fluorescent protein
Adam, Virgile Carpentier, Philippe Violot, Sebastien Lelimousin, Mickaël Darnault, Claudine Nienhaus, G. Ulrich Bourgeois, Dominique # ×
American Chemical Society
Journal of the American Chemical Society vol:131 issue:50 pages:18063-18065
We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of pi conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.