title: Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fluorescent protein.
abstract: 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.
authors: European Synchrotron Radiation Facility, 6 rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France.
authors: Virgile
authors: V
authors: Adam
authors: Philippe
authors: P
authors: Carpentier
authors: Sebastien
authors: S
authors: Violot
authors: Mickaël
authors: M
authors: Lelimousin
authors: Claudine
authors: C
authors: Darnault
authors: G Ulrich
authors: GU
authors: Nienhaus
authors: Dominique
authors: D
authors: Bourgeois
mesh_terms: D018360:Crystallography, X-Ray; D004579:Electron Transport; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000528:radiation effects*; D008956:Models, Chemical; D038761:Photobleaching; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D014965:X-Rays
chemical_list: D049452:Green Fluorescent Proteins