title: Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations.
abstract: Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoconversion. Here, we have combined crystallography and (in crystallo) spectroscopy to characterize the Phe-173-Ser mutant of the tetrameric variant of EosFP, named IrisFP, which incorporates both types of phototransformations. In its green fluorescent state, IrisFP displays reversible photoswitching, which involves cis-trans isomerization of the chromophore. Like its parent protein EosFP, IrisFP also photoconverts irreversibly to a red-emitting state under violet light because of an extension of the conjugated pi-electron system of the chromophore, accompanied by a cleavage of the polypeptide backbone. The red form of IrisFP exhibits a second reversible photoswitching process, which may also involve cis-trans isomerization of the chromophore. Therefore, IrisFP displays altogether 3 distinct photoactivation processes. The possibility to engineer and precisely control multiple phototransformations in photoactivatable FPs offers exciting perspectives for the extension of the fluorescent protein toolkit.
authors.affiliation: European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, BP 220, 38043 Grenoble Cedex, France.
authors.forename: Virgile
authors.initials: V
authors.lastname: Adam
authors.forename: Mickaël
authors.initials: M
authors.lastname: Lelimousin
authors.forename: Susan
authors.initials: S
authors.lastname: Boehme
authors.forename: Guillaume
authors.initials: G
authors.lastname: Desfonds
authors.forename: Karin
authors.initials: K
authors.lastname: Nienhaus
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Field
authors.forename: Joerg
authors.initials: J
authors.lastname: Wiedenmann
authors.forename: Sean
authors.initials: S
authors.lastname: McSweeney
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
authors.forename: Dominique
authors.initials: D
authors.lastname: Bourgeois
mesh_terms: D018360:Crystallography, X-Ray; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D010777:Photochemistry; D011487:Protein Conformation
chemical_list: D008164:Luminescent Proteins