Assembly of photoactive orange carotenoid protein from its domains unravels a carotenoid shuttle mechanism

The photoswitchable orange carotenoid protein (OCP) is indispensable for cyanobacterial photoprotection by quenching phycobilisome fluorescence upon photoconversion from the orange OCP^O to the red OCP^R form. Cyanobacterial genomes frequently harbor, besides genes for orange carotenoid proteins (OCPs), several genes encoding homologs of OCP’s N- or C-terminal domains (NTD, CTD). Unlike the well-studied NTD homologs, called Red Carotenoid Proteins (RCPs), the role of CTD homologs remains elusive. We show how OCP can be reassembled from its functional domains. Expression of Synechocystis OCP-CTD in carotenoid-producing Escherichia coli yielded violet-colored proteins, which, upon mixing with the RCP-apoprotein, produced an orange-like photoswitchable form that further photoconverted into a species that quenches phycobilisome fluorescence and is spectroscopically indistinguishable from RCP, thus demonstrating a unique carotenoid shuttle mechanism. Spontaneous carotenoid transfer also occurs between canthaxanthin-coordinating OCP-CTD and the OCP apoprotein resulting in formation of photoactive OCP. The OCP-CTD itself is a novel, dimeric carotenoid-binding protein, which can coordinate canthaxanthin and zeaxanthin, effectively quenches singlet oxygen and interacts with the Fluorescence Recovery Protein. These findings assign physiological roles to the multitude of CTD homologs in cyanobacteria and explain the evolutionary process of OCP formation.

     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

     

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle

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.     

Thermodynamics of the melting of PNA(2)/DNA triple helices.

Equilibrium melting curves were obtained for triplexes, formed by single stranded DNA containing an A10 target with bis-PNA consisting of two T10 decamers. Thermodynamic parameters of melting were determined for Na(+) concentrations 50, 200 and 600mM by two methods. The melting enthalpy Delta H degrees was evaluated from the width of the differential melting curves and from the concentration dependence of the melting temperature. The latter method allowed also evaluating the melting entropy Delta S degrees. The following results were obtained: Delta H degrees = - 137 kcal/M, Delta S degrees = - 368 cal/M.K, Delta G degrees (298)= - 27 kcal/M. No dependence of Delta H degrees, Delta S degrees and Delta G degrees (298) was observed upon ionic strength within the accuracy of the experiment (+/- 10%). The absolute values of Delta H degrees, Delta S degrees and Delta G degrees(298) are 2 to 3 times higher than the published values of corresponding melting parameters for decameric PNA/DNA duplexes of various nucleic base sequences. The origin of the extremely high stability of the triplexes is discussed.     

Expression, purification and some properties of fluorescent chimeras of human small heat shock proteins

Small heat shock proteins (sHsp) are ubiquitously expressed in all human tissues and have an important housekeeping role in preventing the accumulation of aggregates of improperly folded or denatured proteins. They also participate in the regulation of the cytoskeleton, proliferation, apoptosis and many other vital processes. Fluorescent chimeras composed of sHsp and enhanced fluorescent proteins have been used to determine the intracellular locations of small heat shock proteins and to analyse the hetero-oligomeric complexes formed by different sHsp. However, the biochemical properties and chaperone-like activities of these chimeras have not been investigated. To determine the properties of these chimeras, we fused enhanced yellow and cyan fluorescent proteins (EYFP and ECFP) to the N-termini of four ubiquitously expressed human small heat shock proteins: HspB1, HspB5, HspB6, and HspB8. The eight fluorescent chimeras of small heat shock proteins and isolated fluorescent proteins were expressed in Escherichia coli. The chimeric proteins were isolated and purified via ammonium sulphate fractionation, ion exchange and size-exclusion chromatography. This method provided 20-100 mg of fluorescent chimeras from 1 L of bacterial culture. The spectral properties of the chimeras were similar to those of the isolated fluorescent proteins. The fusion of fluorescent proteins to HspB6 and HspB8, which typically form dimers, did not affect their quaternary structures. Oligomers of the fluorescent chimeras of HspB1 and HspB5 were less stable and contained fewer subunits than oligomers formed by the wild-type proteins. Fusion with EYFP decreased the chaperone-like activity of HspB5 and HspB6 whereas fusion with ECFP increased chaperone-like activity. All fluorescent chimeras of HspB1 and HspB8 had higher chaperone-like activity than the wild-type proteins. Thus, although fluorescent chimeras are useful for many purposes, the fluorescent proteins used to form these chimeras may affect certain important properties of sHsp.     

Radioprotective role of cyanobacterial phycobilisomes

Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called “Great Oxygenation Event” that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.     

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

Serine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.

Structured summary

MINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)MINT-7233285, MINT-7233297, MINT-7233310: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

Does Interaction between the Motor and Regulatory Domains of the Myosin Head Occur during ATPase Cycle? Evidence from Thermal Unfolding Studies on Myosin Subfragment 1

Myosin head (myosin subfragment 1, S1) consists of two major structural domains, the motor (or catalytic) domain and the regulatory domain. Functioning of the myosin head as a molecular motor is believed to involve a rotation of the regulatory domain (lever arm) relative to the motor domain during the ATPase cycle. According to predictions, this rotation can be accompanied by an interaction between the motor domain and the C-terminus of the essential light chain (ELC) associated with the regulatory domain. To check this assumption, we applied differential scanning calorimetry (DSC) combined with temperature dependences of fluorescence to study changes in thermal unfolding and the domain structure of S1, which occur upon formation of the ternary complexes S1-ADP-AlF₄ ^- and S1-ADP-BeF_x that mimic S1 ATPase intermediate states S1**-ADP-P_i and S1*-ATP, respectively. To identify the thermal transitions on the DSC profiles (i.e. to assign them to the structural domains of S1), we compared the DSC data with temperature-induced changes in fluorescence of either tryptophan residues, located only in the motor domain, or recombinant ELC mutants (light chain 1 isoform), which were first fluorescently labeled at different positions in their C-terminal half and then introduced into the S1 regulatory domain. We show that formation of the ternary complexes S1-ADP-AlF₄ ^- and S1-ADP-BeF_x significantly stabilizes not only the motor domain, but also the regulatory domain of the S1 molecule implying interdomain interaction via ELC. This is consistent with the previously proposed concepts and also adds some new interesting details to the molecular mechanism of the myosin ATPase cycle.     

Features of protein−protein interactions in the cyanobacterial photoprotection mechanism

Photoprotective mechanisms of cyanobacteria are characterized by several features associated with the structure of their water-soluble antenna complexes–the phycobilisomes (PBs). During energy transfer from PBs to chlorophyll of photosystem reaction centers, the “energy funnel” principle is realized, which regulates energy flux due to the specialized interaction of the PBs core with a quenching molecule capable of effectively dissipating electron excitation energy into heat. The role of the quencher is performed by ketocarotenoid within the photoactive orange carotenoid protein (OCP), which is also a sensor for light flux. At a high level of insolation, OCP is reversibly photoactivated, and this is accompanied by a sig- nificant change in its structure and spectral characteristics. Such conformational changes open the possibility for pro- tein–protein interactions between OCP and the PBs core (i.e., activation of photoprotection mechanisms) or the fluores- cence recovery protein. Even though OCP was discovered in 1981, little was known about the conformation of its active form until recently, as well as about the properties of homologs of its N and C domains. Studies carried out during recent years have made a breakthrough in understanding of the structural-functional organization of OCP and have enabled discovery of new aspects of the regulation of photoprotection processes in cyanobacteria. This review focuses on aspects of protein–pro- tein interactions between the main participants of photoprotection reactions and on certain properties of representatives of newly discovered families of OCP homologs.