title: Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle.
abstract: Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.
authors.affiliation: Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia. Electronic address: emaksimoff@yandex.ru.
authors.forename: Eugene G
authors.initials: EG
authors.lastname: Maksimov
authors.affiliation: Laboratory of Structural Biochemistry of Proteins, A. N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
authors.forename: Nikolai N
authors.initials: NN
authors.lastname: Sluchanko
authors.affiliation: Laboratory of Cell Regulation, K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Kirill S
authors.initials: KS
authors.lastname: Mironov
authors.affiliation: Institute of Chemistry, Technical University of Berlin, Berlin, Germany.
authors.forename: Evgeny A
authors.initials: EA
authors.lastname: Shirshin
authors.affiliation: Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Konstantin E
authors.initials: KE
authors.lastname: Klementiev
authors.affiliation: Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Georgy V
authors.initials: GV
authors.lastname: Tsoraev
authors.affiliation: Department of Intracellular Regulation, Institute of Plant Physiology, Moscow, Russia.
authors.forename: Marcus
authors.initials: M
authors.lastname: Moldenhauer
authors.affiliation: Department of Intracellular Regulation, Institute of Plant Physiology, Moscow, Russia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
authors.affiliation: Department of Intracellular Regulation, Institute of Plant Physiology, Moscow, Russia.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Los
authors.affiliation: Laboratory of Cell Regulation, K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia; Institute of Basic Biological Problems, Russian Academy of Sciences, Moscow, Russia. Electronic address: suleyman.allakhverdiev@gmail.com.
authors.forename: Suleyman I
authors.initials: SI
authors.lastname: Allakhverdiev
authors.affiliation: Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Vladimir Z
authors.initials: VZ
authors.lastname: Paschenko
authors.affiliation: Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Rubin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D017440:Photoperiod; D011487:Protein Conformation; D012235:Rhodamines / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014783:Viscosity
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D012235:Rhodamines; D012997:Solvents; C109473:orange carotenoid protein, Synechocystis; C005358:tetramethylrhodamine