The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state, OCPR, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCPO suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCPR. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.

The 35 kDa water-soluble Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor that simultaneously serves as efficient quencher of phycobilisome excitation energy as well as of reactive oxygen species. Photoactivation triggers largescale conformational rearrangements to convert OCP from the orange OCP O state to the red active signaling state OCP R , as demonstrated by various structural methods. Eventually, such rearrangements imply complete yet reversible separation of structural domains (C-and N-terminal domain) and significant translocation of the carotenoid cofactor. Very recently, dynamic crystallography of OCP O crystals suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions in the protein. However, the currently existing gap between the ultra-fast picosecond and 100 millisecond time scale of spectroscopic and structural data precludes knowledge about distinct intermediate states. In this study, we took advantage of single 7 ns laser pulses to study peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/167478 doi: bioRxiv preprint first posted online Jul. 23, 2017; 2 carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state OCP R . In addition, time-resolved fluorescence spectroscopy and following assignment of carotenoid-induced quenching of different tryptophan residues revealed a novel orange intermediate state, which appears during back-relaxation of photoactivated OCP R to OCP O . Our results show asynchronous changes in the carotenoid and protein components and provide refined mechanistic information about the OCP photocycle as well as introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.

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The intracellular space of red blood cells (RBC) includes 40% hemoglobin (Hb) molecules and 60% being water molecules. When the intracellular volume changes, which is possible when erythrocyte cells pass through the blood stream, the possibility of redistribution of intracellular components of the cell due to its volume changes is assumed. By methods of optical spectroscopy (non-invasive methods of infrared spectroscopy, Raman-spectroscopy, laser interference microscopy) changes of morphology, conformation and redistribution of Hb have been revealed in the human erythrocyte due to an increase in the ratio of [Na+]in and [K+]in when Na+/K+-ATPase is blocked in the cell. The decrease of activity of Na+/K+-ATPase by a ouabaine (3 mM) has been found to lead not only to an increase in [Na+]in cell but also to an increase in positive charge on the cytoplasmic surface of the RBC membrane. In these conditions, changes in the conformation of both the heme and globine parts of the cytoplasmic Hb have been identified. It has been shown that cell depolarization, together with cell volume change, leads to a decrease in packing density of Hb molecule, which may be related to sorption of intracellular Na+ (or Ca2+) c Hb, as well as increasing the amount of water molecules in the cell and redistributin Hb in the cell. These processes can lead to a change in the conformation of Hb, as well as to a redistribution and alteration of the conformation of the cytoplasmic Hb.