TY - JOUR
T1 - Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle
AU - Maksimov, Eugene G.
AU - Klementiev, Konstantin E.
AU - Tsoraev, Georgy V.
AU - Paschenko, Vladimir Z.
AU - Rubin, Andrei B.
AU - Sluchanko, Nikolai N.
AU - Mironov, Kirill S.
AU - Allakhverdiev, Suleyman I.
AU - Shirshin, Evgeny A.
AU - Moldenhauer, Marcus
AU - Friedrich, Thomas
AU - Los, Dmitry A.
N1 - Funding Information:
This work was supported by grants from the Russian Foundation for Basic Research (RFBR; nos. 14-04-01536a, 15-04-01930a, and 16-34-00394mol_a to V.Z.P., E.G.M., and K.E.K.), the Russian Science Foundation (no. 14-17-00451 to E.A.S. and E.G.M and no. 14-24-00020 to K.S.M. and D.A.L.). E.G.M., G.V.T., and K.E.K. were also supported by the Russian Ministry of Education and Science (no. MK-5949.2015.4). E.G.M. was supported by a Dynasty Foundation Fellowship. The reported study was funded by the RFBR and the Moscow city Government according to research project no. 15-34-70007 ?mol_?_mos?. T.F. acknowledges support from the German Federal Ministry of Education and Research (WTZ-RUS grant 01DJ15007) and the German Research Foundation (Cluster of Excellence ?Unifying Concepts in Catalysis?). S.I.A. was supported by a grant from the Russian Science Foundation (no. 14-14-00039).
Publisher Copyright:
© 2017 Biophysical Society
PY - 2017/1/10
Y1 - 2017/1/10
N2 - 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.
AB - 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.
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U2 - 10.1016/j.bpj.2016.11.3193
DO - 10.1016/j.bpj.2016.11.3193
M3 - Article
C2 - 28076815
AN - SCOPUS:85008646183
VL - 112
SP - 46
EP - 56
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 1
ER -