Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle.

Autor:	Maksimov EG; Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia. Electronic address: emaksimoff@yandex.ru., Sluchanko NN; Laboratory of Structural Biochemistry of Proteins, A. N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia., Mironov KS; Laboratory of Cell Regulation, K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia., Shirshin EA; Institute of Chemistry, Technical University of Berlin, Berlin, Germany., Klementiev KE; Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia., Tsoraev GV; Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia., Moldenhauer M; Department of Intracellular Regulation, Institute of Plant Physiology, Moscow, Russia., Friedrich T; Department of Intracellular Regulation, Institute of Plant Physiology, Moscow, Russia., Los DA; Department of Intracellular Regulation, Institute of Plant Physiology, Moscow, Russia., Allakhverdiev SI; 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., Paschenko VZ; Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia., Rubin AB; Department of Biophysics, Lomonosov Moscow State University, Moscow, Russia.
Jazyk:	angličtina
Zdroj:	Biophysical journal [Biophys J] 2017 Jan 10; Vol. 112 (1), pp. 46-56.
DOI:	10.1016/j.bpj.2016.11.3193
Abstrakt:	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. (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu