title: Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism.
abstract: The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCPOto the red OCPRform. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP's N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Moldenhauer
authors.affiliation: A.N. Bach Institute of Biochemistry, Federal Research Center "Fundamentals of Biotechnology", Russian Academy of Sciences, Moscow, Russian Federation, 119071.|Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation, 119992.
authors.forename: Nikolai N
authors.initials: NN
authors.lastname: Sluchanko
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Buhrke
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation, 119992.
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Zlenko
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Neslihan 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: Franz-Josef
authors.initials: FJ
authors.lastname: Schmitt
authors.affiliation: Institute of Chemistry PC 14, Technical University of Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Hildebrandt
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russian Federation, 119992. emaksimoff@yandex.ru.
authors.forename: Eugene 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. friedrich@chem.tu-berlin.de.
authors.forename: Thomas
authors.identifier: http://orcid.org/0000-0003-2835-2895
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001692:Biological Transport / Q000528:radiation effects; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D008027:Light; D008954:Models, Biological; D000072417:Protein Domains; D015202:Protein Engineering; D055503:Protein Multimerization; D046939:Synechocystis / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C109473:orange carotenoid protein, Synechocystis; D002338:Carotenoids
keywords: Analytical size-exclusion chromatography; Carotenoid shuttle; Cyanobacteria; Mass spectrometry; Orange carotenoid protein; Photoactivity; Photoprotection; Phycobilisome fluorescence quenching; Raman spectroscopy; UV–Vis spectroscopy