title: Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.
abstract: Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPRis converted to OCPOand dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288Amutant as a competent model for the signaling state OCPR(Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288Ahas a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288Amutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288Aor the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität 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, 33 Leninsky prospect, building 1, Moscow, Russian Federation, 119071.|Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1, p. 12, Moscow, Russian Federation, 119992.
authors.forename: Nikolai N
authors.initials: NN
authors.lastname: Sluchanko
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Neslihan N
authors.initials: NN
authors.lastname: Tavraz
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Junghans
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Buhrke
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Willoweit
authors.affiliation: Institut für Chemie Sekr. TC 7, Technische Universität Berlin, Straße des 17. Juni 124, 10623, Berlin, Germany.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Chiappisi
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany.
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Schmitt
authors.affiliation: Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet, CMM L8:01, 17176, Stockholm, Sweden.
authors.forename: Vladana
authors.initials: V
authors.lastname: Vukojević
authors.affiliation: Department of Quantum Electronics, Faculty of Physics, M.V. Lomonosov Moscow State University, Leninskie Gory, Moscow, Russian Federation, 119992.
authors.forename: Evgeny A
authors.initials: EA
authors.lastname: Shirshin
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1, p. 12, Moscow, Russian Federation, 119992.
authors.forename: Vladimir Y
authors.initials: VY
authors.lastname: Ponomarev
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1, p. 12, Moscow, Russian Federation, 119992.
authors.forename: Vladimir Z
authors.initials: VZ
authors.lastname: Paschenko
authors.affiliation: Institut für Chemie Sekr. TC 7, Technische Universität Berlin, Straße des 17. Juni 124, 10623, Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Gradzielski
authors.affiliation: Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, 1, p. 12, Moscow, Russian Federation, 119992.
authors.forename: Eugene G
authors.initials: EG
authors.lastname: Maksimov
authors.affiliation: Institut für Chemie Sekr. PC 14, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany. friedrich@chem.tu-berlin.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D004058:Diffusion; D057446:Hydrodynamics; D013058:Mass Spectrometry; D015203:Reproducibility of Results; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine
keywords: Fluorescein-maleimide; Fluorescence correlation spectroscopy; Fluorescence recovery protein; Mass spectroscopy; Orange carotenoid protein; Site-specific fluorescence labeling