Solution Structures of Two Different FRP-OCP Complexes as Revealed via SEC-SANS.

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results.

X-ray radiolytic labeling reveals the molecular basis of orange carotenoid protein photoprotection and its interactions with fluorescence recovery protein.

In cyanobacterial photoprotection, the orange carotenoid protein (OCP) is photoactivated under excess light conditions and binds to the light-harvesting antenna, triggering the dissipation of captured light energy. In low light, the OCP relaxes to the native state, a process that is accelerated in the presence of fluorescence recovery protein (FRP). Despite the importance of the OCP in photoprotection, the precise mechanism of photoactivation by this protein is not well-understood. Using time-resolved X-ray-mediated in situ hydroxyl radical labeling, we probed real-time solvent accessibility (SA) changes at key OCP residues during photoactivation and relaxation. We observed a biphasic photoactivation process in which carotenoid migration preceded domain dissociation. We also observed a multiphasic relaxation process, with collapsed domain association preceding the final conformational rearrangement of the carotenoid. Using steady-state hydroxyl radical labeling, we identified sites of interaction between the FRP and OCP. In combination, the findings in this study provide molecular-level insights into the factors driving structural changes during OCP-mediated photoprotection in cyanobacteria, and furnish a basis for understanding the physiological relevance of the FRP-mediated relaxation process.

Optimization of expression of orange carotenoid protein in Escherichia coli.

Naturally-occurring orange carotenoid protein (OCP) is synthesized in cyanobacteria and red algae for photoprotection. Holo-OCP can be produced with three plasmids in E. coli, which needs two inducers (arabinose and isopropyl ß-D-thiogalactoside) to initiate two processes: one for generation of carotenoid and the other for generation of apo-OCP, so takes about two days. Afterwards, a two-plasmid method using two plasmids in E. coli is established, in which E. coli cells are induced only by isopropyl ß-D-thiogalactoside, so can yield different holo-OCPs from several cyanobacteria within three days. In this work, we optimized the two-plasmid method as follows: (1) re-organization of the two plasmids, letting carotenoid-generating gene, crtW, be arranged together with apo-OCP-generating gene, ocp, in a single plasmid, which causes that both carotenoid and apo-protein were properly produced, (2) modification of several amino acids at the N-terminus of apo-OCP, in this way increasing the yield and purity of holo-OCP. After these optimizations, we can generate much more amount of holo-OCP within shorter time of only 16 h, and pure holo-OCP be conveniently prepared after routine purification. Comparing with the reported data, the general yield of holo-OCP is increased by ∼10-fold under similar conditions. The high quality of the prepared holo-OCPs is verified by fluorescence quenching of the phycobilisomes.

Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP.

Photosynthesis requires a balance between efficient light harvesting and protection against photodamage. The cyanobacterial photoprotection system uniquely relies on the functioning of the photoactive orange carotenoid protein (OCP) that under intense illumination provides fluorescence quenching of the light-harvesting antenna complexes, phycobilisomes. The recently identified fluorescence recovery protein (FRP) binds to the photoactivated OCP and accelerates its relaxation into the basal form, completing the regulatory circle. The molecular mechanism of FRP functioning is largely controversial. Moreover, since the available knowledge has mainly been gained from studying Synechocystis proteins, the cross-species conservation of the FRP mechanism remains unexplored. Besides phylogenetic analysis, we performed a detailed structural-functional analysis of two selected low-homology FRPs by comparing them with Synechocystis FRP (SynFRP). While adopting similar dimeric conformations in solution and preserving binding preferences of SynFRP towards various OCP variants, the low-homology FRPs demonstrated distinct binding stoichiometries and differentially accentuated features of this functional interaction. By providing clues to understand the FRP mechanism universally, our results also establish foundations for upcoming structural investigations necessary to elucidate the FRP-dependent regulatory mechanism.

Site, trigger, quenching mechanism and recovery of non-photochemical quenching in cyanobacteria: recent updates.

Cyanobacteria exhibit a novel form of non-photochemical quenching (NPQ) at the level of the phycobilisome. NPQ is a process that protects photosystem II (PSII) from possible highlight-induced photo-damage. Although significant advancement has been made in understanding the NPQ, there are still some missing details. This critical review focuses on how the orange carotenoid protein (OCP) and its partner fluorescence recovery protein (FRP) control the extent of quenching. What is and what is not known about the NPQ is discussed under four subtitles; where does exactly the site of quenching lie? (site), how is the quenching being triggered? (trigger), molecular mechanism of quenching (quenching) and recovery from quenching. Finally, a recent working model of NPQ, consistent with recent findings, is been described.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

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.

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.

Structure and function of the light-protective orange carotenoid protein families.

Orange carotenoid proteins (OCPs) are unique photoreceptors that are critical for cyanobacterial photoprotection. Upon exposure to blue-green light, OCPs are activated from a stable orange form, OCPO, to an active red form, OCPR, which binds to phycobilisomes (PBSs) and performs photoprotective non-photochemical quenching (NPQ). OCPs can be divided into three main families: the most abundant and best studied OCP1, and two others, OCP2 and OCP3, which have different activation and quenching properties and are yet underexplored. Crystal structures have been acquired for the three OCP clades, providing a glimpse into the conformational underpinnings of their light-absorption and energy dissipation attributes. Recently, the structure of the PBS-OCPR complex has been obtained allowing for an unprecedented insight into the photoprotective action of OCPs. Here, we review the latest findings in the field that have substantially improved our understanding of how cyanobacteria protect themselves from the toxic consequences of excess light absorption. Furthermore, current research is applying the structure of OCPs to bio-inspired optogenetic tools, to function as carotenoid delivery devices, as well as engineering the NPQ mechanism of cyanobacteria to enhance their photosynthetic biomass production.

Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function.

Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCPO) to the red (OCPR) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCPR accumulation implicates restructuring of a compact dark-adapted OCPO state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCPR-like absorption spectrum and an OCPO-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCPCC). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCPCC, FRP binds reduced OCPCC nearly as efficiently as OCPO devoid of the NTE, suggesting that the low-affinity FRP binding to OCPO is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCPCC indicates that OCPCC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCPR-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.

Deletion of the short N-terminal extension in OCP reveals the main site for FRP binding.

The orange carotenoid protein (OCP) plays a key role in cyanobacterial photoprotection. Photoconversion entails structural rearrangements in OCP that are required for its binding to phycobilisome, thereby inducing excitation energy dissipation. Detachment of OCP from phycobilisome requires the fluorescence recovery protein (FRP). It is considered that OCP interacts with FRP only in the photoactivated state; however, the binding site for FRP is currently unknown. As an important stabilizing element in orange OCP, the short αA-helix within the N-terminal extension (NTE) binds to the C-terminal domain (CTD), but unfolds upon photoactivation and interferes with phycobilisome binding. Here, we demonstrate that the NTE shares specific structural and functional similarities with FRP and discover the main site of OCP-FRP interactions in the OCP-CTD.

Role of inter-domain cavity in the attachment of the orange carotenoid protein to the phycobilisome core and to the fluorescence recovery protein.

Using molecular modeling and known spatial structure of proteins, we have derived a universal 3D model of the orange carotenoid protein (OCP) and phycobilisome (PBS) interaction in the process of non-photochemical PBS quenching. The characteristic tip of the phycobilin domain of the core-membrane linker polypeptide (LCM) forms the attachment site on the PBS core surface for interaction with the central inter-domain cavity of the OCP molecule. This spatial arrangement has to be the most advantageous one because the LCM, as the major terminal PBS-fluorescence emitter, accumulates energy from the most other phycobiliproteins within the PBS before quenching by OCP. In agreement with the constructed model, in cyanobacteria, the small fluorescence recovery protein is wedged in the OCP's central cavity, weakening the PBS and OCP interaction. The presence of another one protein, the red carotenoid protein, in some cyanobacterial species, which also can interact with the PBS, also corresponds to this model.