title: A comparative study of three signaling forms of the orange carotenoid protein.
abstract: Orange carotenoid protein (OCP) is a water-soluble photoactive protein responsible for a photoprotective mechanism of nonphotochemical quenching in cyanobacteria. Under blue-green illumination, OCP converts from the stable orange into the signaling red quenching form; however, the latter form could also be obtained by chemical activation with high concentrations of sodium thiocyanate (NaSCN) or point mutations. In this work, we show that a single replacement of tryptophan-288, normally involved in protein-chromophore interactions, by alanine, results in formation of a new protein form, hereinafter referred to as purple carotenoid protein (PCP). Comparison of resonance Raman spectra of the native photoactivated red form, chemically activated OCP, and PCP reveals that carotenoid conformation is sensitive to the structure of the C-domain, implicating that the chromophore retains some interactions with this part of the protein in the active red form. Combination of differential scanning fluorimetry and picosecond time-resolved fluorescence anisotropy measurements allowed us to compare the stability of different OCP forms and to estimate relative differences in protein rotation rates. These results were corroborated by hydrodynamic analysis of proteins by dynamic light scattering and analytical size-exclusion chromatography, indicating that the light-induced conversion of the protein is accompanied by a significant increase in its size. On the whole, our data support the idea that the red form of OCP is a molten globule-like protein in which, however, interactions between the carotenoid and the C-terminal domain are preserved.
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia. emaksimoff@yandex.ru.
authors.forename: E G
authors.initials: EG
authors.lastname: Maksimov
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Moldenhauer
authors.affiliation: Department of Quantum Electronics, Faculty of Physics, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: E A
authors.initials: EA
authors.lastname: Shirshin
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: E A
authors.initials: EA
authors.lastname: Parshina
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia.
authors.forename: N N
authors.initials: NN
authors.lastname: Sluchanko
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: K E
authors.initials: KE
authors.lastname: Klementiev
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: G V
authors.initials: GV
authors.lastname: Tsoraev
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: N N
authors.initials: NN
authors.lastname: Tavraz
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Willoweit
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: F-J
authors.initials: FJ
authors.lastname: Schmitt
authors.affiliation: Institute for Molecular Biosciences, Johann Wolfgang Goethe University of Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Breitenbach
authors.affiliation: Institute for Molecular Biosciences, Johann Wolfgang Goethe University of Frankfurt, Max-von-Laue-Straße 9, 60438, Frankfurt, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Sandmann
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: V Z
authors.initials: VZ
authors.lastname: Paschenko
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Friedrich
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia.
authors.forename: A B
authors.initials: AB
authors.lastname: Rubin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000502:physiology; D005453:Fluorescence; D005454:Fluorescence Polarization; D005470:Fluorometry; D013059:Spectrum Analysis, Raman; D046939:Synechocystis / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; C109473:orange carotenoid protein, Synechocystis
keywords: Echinenone; Fluorescence anisotropy; Fluorescence lifetime; Orange carotenoid protein; Protein expression; Synechocystis sp. PCC 6803