Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

Maksimov, E.G.; Sluchanko, N.N.; Mironov, K.S.; Shirshin, E.A.; Klementiev, K.E.; Tsoraev, G.V.; Moldenhauer, M.; Friedrich, T.; Los, D.A.; Allakhverdiev, S.I.; Paschenko, V.Z.; Rubin, A.B.

Авторы: Maksimov E.G., Sluchanko N.N., Mironov K.S., Shirshin E.A., Klementiev K.E., Tsoraev G.V., Moldenhauer M., Friedrich T., Los D.A., Allakhverdiev S.I., Paschenko V.Z., Rubin A.B.
Журнал: Biophysical Journal
Том: 112
Год издания: 2017
Издательство: Biophysical Society
Местоположение издательства: United States
Первая страница: 46
Последняя страница: 56
DOI: 10.1016/j.bpj.2016.11.3193
Аннотация: Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatusunder excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stableorange to the signaling red state. This is thought to involve a 12-A° translocation of the carotenoid cofactor and separation of theN- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion andassociated 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 distancesfrom 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 Fo¨ rster 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 A° , 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 conversionof 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.
Добавил в систему: Пащенко Владимир Захарович

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Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycleстатья

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