Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fluorescent protein.

Virgile Adam (1, 2) , Philippe Carpentier (3) , Sebastien Violot (4) , Mickaël Lelimousin (3) , Claudine Darnault (3) , G Ulrich Nienhaus (5, 6) , Dominique Bourgeois (3)

1 IBS - UMR 5075 - Institut de biologie structurale
2 ESRF - European Synchrotron Radiation Facility [Grenoble]
3 IBS - UMR 5075 - Institut de biologie structurale
4 IRTSV - Institut de Recherches en Technologies et Sciences pour le Vivant
5 KIT - Karlsruhe Institute of Technology
6 Department of Physics, University of Illinois

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.

Mots clés: fluorescent protein; marker; fluorescence; cell biology; photobleaching

Domaines: Biologie cellulaire; Chimie théorique et/ou physique

Virgile Adam, Philippe Carpentier, Sebastien Violot, Mickaël Lelimousin, Claudine Darnault, et al.. Structural basis of X-ray-induced transient photobleaching in a photoactivatable green fluorescent protein.. Journal of the American Chemical Society, 2009, 131 (50), pp.18063-5. ⟨10.1021/ja907296v⟩. ⟨hal-00474158⟩