Chemical activation of the cyanobacterial orange carotenoid protein

The effects of the Hofmeister series of ions on the activation of the orange carotenoid protein (OCP) from the inactive orange form to the active red form were tested. Kosmotropes led to lower OCP activation, whereas chaotropes led to greater OCP activation. Concentrations of thiocyanate exceeding 1.5 M dark activate the orange carotenoid protein to its red form. This chemically activated OCP was studied by UV–vis and circular dichroism spectroscopies. The chemically-activated OCP quenches the fluorescence of phycobilisomes in vitro, to a level comparable to that of the light-activated OCP.     

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 (OCP^O) into the red-shifted, active state (OCP^R) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCP^R is converted to OCP^O and dissociates from PBs; however, the mode and site of OCP^R/FRP interactions remain elusive. Recently, we have introduced the purple OCP^W288A mutant as a competent model for the signaling state OCP^R (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 OCP^W288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCP^O, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCP^O, a substantial compaction of the OCP^W288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCP^W288A 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.     

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 (L_CM) 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 L_CM, 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.     

Engineering the orange carotenoid protein for applications in synthetic biology

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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.

     

Spectroscopic properties of the carotenoid 3'-hydroxyechinenone in the orange carotenoid protein from the cyanobacterium Arthrospira maxima

The cyanobacterial water-soluble orange carotenoid binding protein (OCP) is an ideal system for study of the effects of protein environment on photophysical properties of carotenoids. It contains a single pigment, the carotenoid 3'-hydoxyechinenone (hECN). In this study, we focus on spectroscopic properties of hECN in solution and in the OCP, aiming to elucidate the spectroscopic effects of the carotenoid-protein interaction in the context of the function(s) of the OCP. The noncovalent binding of hECN to the OCP causes a conformational change in the hECN, leading to a prolongation of the effective conjugation length. This change is responsible for shortening of the S(1) lifetime from 6.5 ps in solution to 3.3 ps in the OCP. The conformational change and the hydrogen bonding via the carbonyl group of hECN result in stabilization of an intramolecular charge-transfer (ICT) state. No signs of the ICT state were found in hECN in solution, regardless of the solvent polarity; spectral bands in transient absorption spectra of OCP-bound hECN exhibit features typical for the ICT state. Application of global fitting analysis revealed further effects of binding hECN in the OCP. The S(1) state of hECN in the OCP decays with two time constants of 0.9 and 3.3 ps. Modeling of the excited-state processes suggests that these two components are due to two populations of hECN in the OCP that differ in the hydrogen bonding via the carbonyl group. These results support the hypothesis that the OCP functions as a photoprotective shield under excess light. Mechanistically, the broadening of the hECN absorption spectrum upon binding to OCP enhances filtering effect of hECN. Furthermore, the binding-induced conformational change and activation of the ICT state that leads to a shortening of hECN lifetime effectively makes the protein-bound hECN a more effective energy dissipator.     

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 (OCP^O) to the red (OCP^R) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCP^R accumulation implicates restructuring of a compact dark-adapted OCP^O 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 OCP^R-like absorption spectrum and an OCP^O-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 (OCP_CC). 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 OCP_CC, FRP binds reduced OCP_CC nearly as efficiently as OCP^O devoid of the NTE, suggesting that the low-affinity FRP binding to OCP^O is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCP_CC indicates that OCP_CC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCP^R-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.     

The orange carotenoid protein in photoprotection of photosystem II in cyanobacteria

Photoprotective mechanisms have evolved in photosynthetic organisms to cope with fluctuating light conditions. Under high irradiance, the production of dangerous oxygen species is stimulated and causes photo-oxidative stress. One of these photoprotective mechanisms, non photochemical quenching (qE), decreases the excess absorbed energy arriving at the reaction centers by increasing thermal dissipation at the level of the antenna. In this review we describe results leading to the discovery of this process in cyanobacteria (qE(cya)), which is mechanistically distinct from its counterpart in plants, and recent progress in the elucidation of this mechanism. The cyanobacterial photoactive soluble orange carotenoid protein is essential for the triggering of this photoprotective mechanism. Light induces structural changes in the carotenoid and the protein leading to the formation of a red active form. The activated red form interacts with the phycobilisome, the cyanobacterial light-harvesting antenna, and induces a decrease of the phycobilisome fluorescence emission and of the energy arriving to the reaction centers. The orange carotenoid protein is the first photoactive protein to be identified that contains a carotenoid as the chromophore. Moreover, its photocycle is completely different from those of other photoactive proteins. A second protein, called the Fluorescence Recovery Protein encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in dislodging the red orange carotenoid protein from the phycobilisome and in the conversion of the free red orange carotenoid protein to the orange, inactive, form. This protein is essential to recover the full antenna capacity under low light conditions after exposure to high irradiance. This article is part of a Special Issue entitled: Photosystem II.     

Occurrence and function of the orange carotenoid protein in photoprotective mechanisms in various cyanobacteria

Excess light is harmful for photosynthetic organisms. The cyanobacterium Synechocystis PCC 6803 protects itself by dissipating the excess of energy absorbed by the phycobilisome, the water-soluble antenna of Photosystem II, into heat decreasing the excess energy arriving to the reaction centers. Energy dissipation results in a detectable decrease of fluorescence. The soluble Orange Carotenoid Protein (OCP) is essential for this blue-green light induced mechanism. OCP genes appear to be highly conserved among phycobilisome-containing cyanobacteria with few exceptions. Here, we show that only the strains containing a whole OCP gene can perform a blue-light induced photoprotective mechanism under both iron-replete and iron-starvation conditions. In contrast, strains containing only N-terminal and/or C-terminal OCP-like genes, or no OCP-like genes at all lack this light induced photoprotective mechanism and they were more sensitive to high-light illumination. These strains must adopt a different strategy to longer survive under stress conditions. Under iron starvation, the relative decrease of phycobiliproteins was larger in these strains than in the OCP-containing strains, avoiding the appearance of a population of dangerous, functionally disconnected phycobilisomes. The OCP-containing strains protect themselves from high light, notably under conditions inducing the appearance of disconnected phycobilisomes, using the energy dissipation OCP-phycobilisome mechanism.     

Further characterization of the orange carotenoid protein extracted from the marine demosponge Axinella verrucosa

The orange coloration of the marine sponge A. verrucosa is provided by some carotenoids widespread in the ectosome and mesohyl of this sponge. These carotenoids are bound to a glyco(lipo)protein forming a non covalent complex. Six carotenoids are bound to the glyco(lipo)protein, but only alpha-carotene was identified by HPLC. The aminoacid composition is quite different from those previously reported in Porifera. The content of Ser and Gly and the total polar residues are high. The presence of Met and Pro was not evidenced. Some unusual aminoacids were detected, of which only Taurine was probably identified.     

Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion

Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas. This regulation permits the cell to meet its cellular energy requirements and at the same time protects the photosynthetic apparatus under fluctuating light conditions. Several reports have revealed that thermal dissipation increases under excess copper in plants. To explore the effects and mechanisms of copper on cyanobacteria NPQ, photoactivation and relaxation of OCP in the presence of copper were examined in this communication. When OCP^o (OCP at orange state) is converted into OCP^r(OCP at red state), copper ion has no effect on the photoactivation kinetics. Relaxation of OCP^r to OCP^o, however, is largely delayed—almost completely blocked, in the presence of copper. Even the addition of the fluorescence recovery protein (FRP) cannot activate the relaxation process. Native polyacrylamide gel electrophoresis (PAGE) analysis result indicates the heterogeneous population of Cu²⁺-locked OCP^r. The Cu²⁺-OCP binding constant was estimated using a hyperbolic binding curve. Functional roles of copper-binding OCP in vivo are discussed.     

Ultrafast spectroscopy tracks carotenoid configurations in the orange and red carotenoid proteins from cyanobacteria

A quenching mechanism mediated by the orange carotenoid protein (OCP) is one of the ways cyanobacteria protect themselves against photooxidative stress. Here, we present a femtosecond spectroscopic study comparing OCP and RCP (red carotenoid protein) samples binding different carotenoids. We confirmed significant changes in carotenoid configuration upon OCP activation reported by Leverenz et al. (Science 348:1463–1466. doi:  10.1126/science.aaa7234, 2015) by comparing the transient spectra of OCP and RCP. The most important marker of these changes was the magnitude of the transient signal associated with the carotenoid intramolecular charge-transfer (ICT) state. While OCP with canthaxanthin exhibited a weak ICT signal, it increased significantly for canthaxanthin bound to RCP. On the contrary, a strong ICT signal was recorded in OCP binding echinenone excited at the red edge of the absorption spectrum. Because the carbonyl oxygen responsible for the appearance of the ICT signal is located at the end rings of both carotenoids, the magnitude of the ICT signal can be used to estimate the torsion angles of the end rings. Application of two different excitation wavelengths to study OCP demonstrated that the OCP sample contains two spectroscopically distinct populations, none of which is corresponding to the photoactivated product of OCP.     

Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism

Plants and algae have developed multiple protective mechanisms to survive under high light conditions. Thermal dissipation of excitation energy in the membrane-bound chlorophyll-antenna of photosystem II (PSII) decreases the energy arriving at the reaction center and thus reduces the generation of toxic photo-oxidative species. This process results in a decrease of PSII-related fluorescence emission, known as non-photochemical quenching (NPQ). It has always been assumed that cyanobacteria, the progenitor of the chloroplast, lacked an equivalent photoprotective mechanism. Recently, however, evidence has been presented for the existence of at least three distinct mechanisms for dissipating excess absorbed energy in cyanobacteria. One of these mechanisms, characterized by a blue-light-induced fluorescence quenching, is related to the phycobilisomes, the extramembranal antenna of cyanobacterial PSII. In this photoprotective mechanism the soluble carotenoid-binding protein (OCP) encoded by the slr1963 gene in Synechocystis sp. PCC 6803, of previously unknown function, plays an essential role. The amount of energy transferred from the phycobilisomes to the photosystems is reduced and the OCP acts as the photoreceptor and as the mediator of this antenna-related process. These are novel roles for a soluble carotenoid protein.     

Mass spectrometry footprinting reveals the structural rearrangements of cyanobacterial orange carotenoid protein upon light activation

The orange carotenoid protein (OCP), a member of the family of blue light photoactive proteins, is required for efficient photoprotection in many cyanobacteria. Photoexcitation of the carotenoid in the OCP results in structural changes within the chromophore and the protein to give an active red form of OCP that is required for phycobilisome binding and consequent fluorescence quenching. We characterized the light-dependent structural changes by mass spectrometry-based carboxyl footprinting and found that an α helix in the N-terminal extension of OCP plays a key role in this photoactivation process. Although this helix is located on and associates with the outside of the β-sheet core in the C-terminal domain of OCP in the dark, photoinduced changes in the domain structure disrupt this interaction. We propose that this mechanism couples light-dependent carotenoid conformational changes to global protein conformational dynamics in favor of functional phycobilisome binding, and is an essential part of the OCP photocycle.     

FTIR studies of the photoactivation processes in squid retinochrome

Retinochrome is a photoisomerase of the invertebrate visual system, which converts all-trans-retinal to the 11-cis configuration and supplies it to visual rhodopsin. In this paper, we studied light-induced structural changes in squid retinochrome by means of low-temperature UV-visible and Fourier transform infrared (FTIR) spectroscopy. In PC liposomes, lumi-retinochrome was stable in the wide temperature range between 77 and 230 K. High thermal stability of the primary intermediate in retinochrome is in contrast to the case in rhodopsins. FTIR spectroscopy suggested that the chromophore of lumi-retinochrome is in a relaxed planar 11-cis form, being consistent with its high thermal stability. The chromophore binding pocket of retinochrome appears to accommodate both all-trans and 11-cis forms without a large distortion, and limited protein structural changes between all-trans and 11-cis chromophores may be suitable for the function of retinochrome as a photoisomerase. The analysis of N-D and O-D stretching vibrations in D(2)O revealed that the hydrogen bond of the Schiff base is weaker in retinochrome than in bovine rhodopsin and bacteriorhodopsin, while retinochrome has a water molecule under strongly hydrogen-bonded conditions (O-D stretch at 2334 cm(-)(1)). The hydrogen bond of the water is further strengthened in lumi-retinochrome. The formation of meta-retinochrome accompanies deprotonation of the Schiff base, together with the peptide backbone alterations of alpha-helices, and possible formation of beta-sheets. It was found that the Schiff base proton is not transferred to its counterion, Glu181, but directly released to the aqueous phase in PC liposomes (pH 7.5). This suggests that the Schiff base environment is exposed to solvent in meta-retinochrome, which may be advantageous for the hydrolysis reaction of the Schiff base in the transport of 11-cis-retinal to its shuttle protein.     

Oligomerization of the chromatin-structuring protein H-NS

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.