Georgy V. Tsoraev

The purple Trp288Ala mutant of Synechocystis OCP persistently quenches phycobilisome fluorescence and tightly interacts with FRP.

In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCPR, which is obtained from the stable dark-adapted orange form, OCPO, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCPW288A) is a fully functional analogue of the OCPR form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCPW288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCPW288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.

Membrane fluidity controls redox-regulated cold stress responses in cyanobacteria

Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

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.

Using a 120-cm cyclotron to study the synchronous effects of ionizing radiation and hypomagnetic conditions on the simplest biological objects

The effect of α-particles on one of the simplest unicellular organisms, Synechocystis sp. PCC 6803 cyanobacteria, is studied using a 120-cm cyclotron. Suspensions of cells are irradiated in a cuvette with walls made of thin mylar films. The energy of α-particles in the solution is 24 MeV. The linear energy transfer (LET) of α-particles is close to that of relativistic nuclei of the neon-magnesium group in galactic cosmic rays, making it possible to simulate their effect on biological objects. A number of apparent changes in the Synechocystis spectral characteristics are found for samples irradiated in and outside a hypomagnetic field.

The nature of anomalous temperature dependence of the fluorescence lifetime of allophycocyanin

86 The study of spectral and temporal characteristics of allophycocyanin fluorescence showed that slow freezing is accompanied by an anomalously sharp reduction in the fluorescence lifetime, which is accompanied by an increase in the halffwidth of the fluorescence spectrum. Probably, the nature of the phenomena observed during slow freezing is associi ated with reversible changes in protein structure that lead to a change in the conformation of the chroo mophore phycocyanobilin. Phycobiliproteins (PBP) play the role of light harr vesters in phycobilisomes, antennal complexes of cyanobacteria and red algae, absorbing light and effii ciently transferring the energy of electronic excitation to the photoactive reaction centers. The use of phycoo biliproteins allows cyanobacteria to increase the effii ciency of light harvesting by the reaction centers by an order of magnitude [1–3]. The molecules of all phycoo biliproteins consist of α and βpolypeptide subunits with molecular weights of 16 and 18 kDa, respectively, in the 1 : 1 ratio [1]. The number of chromophores in the (αβ) 1 monomer corresponds to the division of phycobiliproteins into classes. For example, (αβ) 1 allophycocyanin monomer contains two chroo mophores (phycocyanobilins) [4]. Phycocyanobilins are classified with unclosed or linear tetrapyrroles, whose chemical structure is based on four pyrrole rings connected by three monocarbon bridges. Phycobilipp roteins exhibit a welllexpressed ability to aggregate. In nondenaturing conditions in solutions, it is impossible to separately obtain α and βpolypeptides of phycoo biliproteins, which form (αβ) 1 monomers even at very low concentrations. Spontaneous aggregation does not terminate on monomers; at protein concentraa tions higher than 10 –6 M solutions contain predomii nantly (αβ) 3 trimers and (αβ) 6 hexamers of phycobill iproteins [4]. Phycobiliproteins are convenient objects for designing hybrid structures consisting of natural pigment–protein complexes and semiconductor nanocrystals [5]. A special place among phycobiliproteins is occuu pied by allophycocyanins, the structure and photoo physical properties of which provide for effective capp ture of excitation energy from the shorttwave forms of phycobiliproteins (phycocyanins and phycoerythrins) [1, 4]. At the same time, allophycocyanins interact with orange carotenoid protein [6]. As a result, the excitation energy is transferred from phycobiliproteins to the terminal emitters and chlorophylls of reaction centers [7]. Allophycocyanin trimers have a complex structure of absorption spectra, whose shape depends on the temperature. The main maximum in the absorption spectrum (650 nm) is believed to be associated with the formation of trimers and the interaction of chroo mophores …

Radioprotective role of cyanobacterial phycobilisomes

It is now generally accepted that cyanobacteria are responsible for production of oxygen, which led to the so-called “Great Oxygenation Event”. Appearance of dioxygen in Earth’s atmosphere resulted in formation of the ozone layer and the ionosphere, which caused significant reduction of ionizing radiation levels at the surface of our planet. This event not only increased biological diversity but also canceled the urgency of previously developed mechanisms of DNA protection, which allowed to survive and develop in harsh environmental conditions including exposure to cosmic rays. In order to test the hypothesis if one of the oldest organisms on Earth retained ancient protection mechanisms, we studied the effect of ionizing radiation (IoR, here: α-particles with a kinetic energy of about 30 MeV) and space flight during the mission of the Foton-M4 satellite on cells of Synechocystis sp. PCC6803. By analyzing spectral and functional characteristics of photosynthetic membranes we revealed numerous similarities between cells exposed to IoR and after the space mission. In both cases, we found that excitation energy transfer from phycobilisomes to photosystems was interrupted and the concentration of phycobiliproteins was significantly reduced. Although photosynthetic activity was severely suppressed, the effect was reversible and the cells were able to rapidly recover from stress under normal conditions. Moreover, in vitro experiments demonstrated that the effect of IoR on isolated phycobilisomes was completely different from such in vivo. These observations suggest that the actual existence and the uncoupling of phycobilisomes under irradiation stress could play specific role not only in photo-, but also in radioprotection, which was crucial for early stages of evolution and the development of Life on Earth.