The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction.

To deal with fluctuating light condition, cyanobacteria have developed a photoprotective mechanism which, under high light conditions, decreases the energy arriving at the photochemical centers. It relies on a photoswitch, the Orange Carotenoid Protein (OCP). Once photoactivated, OCP binds to the light harvesting antenna, the phycobilisome (PBS), and triggers the thermal dissipation of the excess energy absorbed. Deactivation of the photoprotective mechanism requires the intervention of a third partner, the Fluorescence Recovery Protein (FRP). FRP by interacting with the photoactivated OCP accelerates its conversion to the non-active form and its detachment from the phycobilisome. We have studied the interaction of FRP with free and phycobilisome-bound OCP. Several OCP variants were constructed and characterized. In this article we show that OCP amino acid F299 is essential and D220 important for OCP deactivation mediated by FRP. Mutations of these amino acids did not affect FRP activity as helper to detach OCP from phycobilisomes. In addition, while mutated R60L FRP is inactive on OCP deactivation, its activity on the detachment of the OCP from the phycobilisomes is not affected. Thus, our results demonstrate that FRP has two distinct activities: it accelerates OCP detachment from phycobilisomes and then it helps deactivation of the OCP. They also suggest that different OCP and FRP amino acids could be involved in these two activities.

Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion.

The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30kDa, a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg2+ is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

An easily reversible structural change underlies mechanisms enabling desert crust cyanobacteria to survive desiccation.

Biological desert sand crusts are the foundation of desert ecosystems, stabilizing the sands and allowing colonization by higher order organisms. The first colonizers of the desert sands are cyanobacteria. Facing the harsh conditions of the desert, these organisms must withstand frequent desiccation-hydration cycles, combined with high light intensities. Here, we characterize structural and functional modifications to the photosynthetic apparatus that enable a cyanobacterium, Leptolyngbya sp., to thrive under these conditions. Using multiple in vivo spectroscopic and imaging techniques, we identified two complementary mechanisms for dissipating absorbed energy in the desiccated state. The first mechanism involves the reorganization of the phycobilisome antenna system, increasing excitonic coupling between antenna components. This provides better energy dissipation in the antenna rather than directed exciton transfer to the reaction center. The second mechanism is driven by constriction of the thylakoid lumen which limits diffusion of plastocyanin to P700. The accumulation of P700(+) not only prevents light-induced charge separation but also efficiently quenches excitation energy. These protection mechanisms employ existing components of the photosynthetic apparatus, forming two distinct functional modes. Small changes in the structure of the thylakoid membranes are sufficient for quenching of all absorbed energy in the desiccated state, protecting the photosynthetic apparatus from photoinhibitory damage. These changes can be easily reversed upon rehydration, returning the system to its high photosynthetic quantum efficiency.

Regulation of Orange Carotenoid Protein Activity in Cyanobacterial Photoprotection.

Plants, algae, and cyanobacteria have developed mechanisms to decrease the energy arriving at reaction centers to protect themselves from high irradiance. In cyanobacteria, the photoactive Orange Carotenoid Protein (OCP) and the Fluorescence Recovery Protein are essential elements in this mechanism. Absorption of strong blue-green light by the OCP induces carotenoid and protein conformational changes converting the orange (inactive) OCP into a red (active) OCP. Only the red orange carotenoid protein (OCP(r)) is able to bind to phycobilisomes, the cyanobacterial antenna, and to quench excess energy. In this work, we have constructed and characterized several OCP mutants and focused on the role of the OCP N-terminal arm in photoactivation and excitation energy dissipation. The N-terminal arm largely stabilizes the closed orange OCP structure by interacting with its C-terminal domain. This avoids photoactivation at low irradiance. In addition, it slows the OCP detachment from phycobilisomes by hindering fluorescence recovery protein interaction with bound OCP(r). This maintains thermal dissipation of excess energy for a longer time. Pro-22, at the beginning of the N-terminal arm, has a key role in the correct positioning of the arm in OCP(r), enabling strong OCP binding to phycobilisomes, but is not essential for photoactivation. Our results also show that the opening of the OCP during photoactivation is caused by the movement of the C-terminal domain with respect to the N-terminal domain and the N-terminal arm.

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria.

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity.

Specificity of the cyanobacterial orange carotenoid protein: influences of orange carotenoid protein and phycobilisome structures.

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome (PB), the extramembranous light-harvesting antenna. This mechanism is triggered by the photoactive Orange Carotenoid Protein (OCP), which acts both as the photosensor and the energy quencher. The OCP binds the core of the PB. The structure of this core differs in diverse cyanobacterial strains. Here, using two isolated OCPs and four classes of PBs, we demonstrated that differences exist between OCPs related to PB binding, photoactivity, and carotenoid binding. Synechocystis PCC 6803 (hereafter Synechocystis) OCP, but not Arthrospira platensis PCC 7345 (hereafter Arthrospira) OCP, can attach echinenone in addition to hydroxyechinenone. Arthrospira OCP binds more strongly than Synechocystis OCP to all types of PBs. Synechocystis OCP can strongly bind only its own PB in 0.8 m potassium phosphate. However, if the Synechocystis OCP binds to the PB at very high phosphate concentrations (approximately 1.4 m), it is able to quench the fluorescence of any type of PB, even those isolated from strains that lack the OCP-mediated photoprotective mechanism. Thus, the determining step for the induction of photoprotection is the binding of the OCP to PBs. Our results also indicated that the structure of PBs, at least in vitro, significantly influences OCP binding and the stabilization of OCP-PB complexes. Finally, the fact that the OCP induced large fluorescence quenching even in the two-cylinder core of Synechococcus elongatus PBs strongly suggested that OCP binds to one of the basal allophycocyanin cylinders.

Metabolite Quantification by Fourier Transform Infrared Spectroscopy in Diatoms: Proof of Concept on Phaeodactylum tricornutum.

Diatoms are feedstock for the production of sustainable biocommodities, including biofuel. The biochemical characterization of newly isolated or genetically modified strains is seminal to identify the strains that display interesting features for both research and industrial applications. Biochemical quantification of organic macromolecules cellular quotas are time-consuming methodologies which often require large amount of biological sample. Vibrational spectroscopy is an essential tool applied in several fields of research. A Fourier transform infrared (FTIR) microscopy-based imaging protocol was developed for the simultaneous cellular quota quantification of lipids, carbohydrates, and proteins of the diatom Phaeodactylum tricornutum. The low amount of sample required for the quantification allows the high throughput quantification on small volume cultures. A proof of concept was performed (1) on nitrogen-starved experimental cultures and (2) on three different P. tricornutum wild-type strains. The results are supported by the observation in situ of lipid droplets by confocal and brightfield microscopy. The results show that major differences exist in the regulation of lipid metabolism between ecotypes of P. tricornutum.

DnaK3 Is Involved in Biogenesis and/or Maintenance of Thylakoid Membrane Protein Complexes in the Cyanobacterium Synechocystis sp. PCC 6803.

DnaK3, a highly conserved cyanobacterial chaperone of the Hsp70 family, binds to cyanobacterial thylakoid membranes, and an involvement of DnaK3 in the biogenesis of thylakoid membranes has been suggested. As shown here, light triggers synthesis of DnaK3 in the cyanobacterium Synechocystis sp. PCC 6803, which links DnaK3 to the biogenesis of thylakoid membranes and to photosynthetic processes. In a DnaK3 depleted strain, the photosystem content is reduced and the photosystem II activity is impaired, whereas photosystem I is regular active. An impact of DnaK3 on the activity of other thylakoid membrane complexes involved in electron transfer is indicated. In conclusion, DnaK3 is a versatile chaperone required for biogenesis and/or maintenance of thylakoid membrane-localized protein complexes involved in electron transfer reactions. As mentioned above, Hsp70 proteins are involved in photoprotection and repair of PS II in chloroplasts.

The Fusion Activity of IM30 Rings Involves Controlled Unmasking of the Fusogenic Core.

The inner membrane-associated protein of 30 kDa (IM30, also known as Vipp1) is required for thylakoid membrane biogenesis and maintenance in cyanobacteria and chloroplasts. The protein forms large rings of ∼2 MDa and triggers membrane fusion in presence of Mg2+. Based on the here presented observations, IM30 rings are built from dimers of dimers, and formation of these tetrameric building blocks is driven by interactions of the central coiled-coil, formed by helices 2 and 3, and stabilized via additional interactions mainly involving helix 1. Furthermore, helix 1 as well as C-terminal regions of IM30 together negatively regulate ring-ring contacts. We propose that IM30 rings represent the inactive form of IM30, and upon binding to negatively charged membrane surfaces, the here identified fusogenic core of IM30 rings eventually interacts with the lipid bilayer, resulting in membrane destabilization and membrane fusion. Unmasking of the IM30 fusogenic core likely is controlled by Mg2+, which triggers rearrangement of the IM30 ring structure.

Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time-Resolved Fourier Transform Infrared Spectroscopy.

The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed α-helix (negative 1651 cm-1 band) is observed. At longer times, a 1644 cm-1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of-at least-two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.

Biosynthesis of soluble carotenoid holoproteins in Escherichia coli.

Carotenoids are widely distributed natural pigments that are excellent antioxidants acting in photoprotection. They are typically solubilized in membranes or attached to proteins. In cyanobacteria, the photoactive soluble Orange Carotenoid Protein (OCP) is involved in photoprotective mechanisms as a highly active singlet oxygen and excitation energy quencher. Here we describe a method for producing large amounts of holo-OCP in E.coli. The six different genes involved in the synthesis of holo-OCP were introduced into E. coli using three different plasmids. The choice of promoters and the order of gene induction were important: the induction of genes involved in carotenoid synthesis must precede the induction of the ocp gene in order to obtain holo-OCPs. Active holo-OCPs with primary structures derived from several cyanobacterial strains and containing different carotenoids were isolated. This approach for rapid heterologous synthesis of large quantities of carotenoproteins is a fundamental advance in the production of antioxidants of great interest to the pharmaceutical and cosmetic industries.

PHOTOSYNTHESIS. A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection.

Pigment-protein and pigment-pigment interactions are of fundamental importance to the light-harvesting and photoprotective functions essential to oxygenic photosynthesis. The orange carotenoid protein (OCP) functions as both a sensor of light and effector of photoprotective energy dissipation in cyanobacteria. We report the atomic-resolution structure of an active form of the OCP consisting of the N-terminal domain and a single noncovalently bound carotenoid pigment. The crystal structure, combined with additional solution-state structural data, reveals that OCP photoactivation is accompanied by a 12 angstrom translocation of the pigment within the protein and a reconfiguration of carotenoid-protein interactions. Our results identify the origin of the photochromic changes in the OCP triggered by light and reveal the structural determinants required for interaction with the light-harvesting antenna during photoprotection.