Konstantin E. Klementiev

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.

Anomalous temperature dependence of the fluorescence lifetime of phycobiliproteins

Using a single photon counting technique we have investigated fluorescence decay spectra of phycobiliproteins with picosecond time resolution. The studies were performed in a wide range of temperatures?from 4 to 300?K. Comparing the fluorescence decay kinetics of samples rapidly frozen in liquid nitrogen with samples that were frozen slowly revealed that the temperature-dependent changes of phycobiliproteins fluorescence lifetime reflect the presence of three different stages, with a phase transition between 273 and 263?K that strongly depends on the rate of freezing. When the temperature decreases from 300 to 273?K, the fluorescence lifetime increases from 1.6 to 1.8?ns. In the region from 273 to 263?K we observed a decrease of the fluorescence lifetime, which strongly depends on the freezing rate: a slight decrease at high freezing rate and a drop down to 200?ps lifetime at slow freezing rate. In the low-temperature regime from 263 to 4?K a linear increase in the fluorescence lifetime was observed for all samples. It was found that the strong temperature dependence of the phycobiliprotein fluorescence, especially in the range between 263 and 273?K, is due to the interaction of the solvent with the chromophore bound to the protein. This feature is explained by a photoisomerization of the phycobiliproteins into a quenching form which is naturally prevented by the protein environment. The formation of ice microcrystals at low freezing rate eliminates this ?protective? effect of the protein environment.

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.

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.