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.Full sunlight is dangerous for plants, algae, and cyanobacteria. It can cause oxidative damages leading to the destruction of the photosynthetic apparatus and to cell death. A short-term photoprotective mechanism developed by oxygenic photosynthetic organisms is the reduction of excitation energy being funneled into the photochemical reaction centers by dissipating excess energy as heat at the level of the antennae (Niyogi and Truong, 2013). In plants and green algae, this mechanism involves the membrane chlorophyll antennae, the light-harvesting complex (for review, see Horton et al., 1996; Horton and Ruban, 2005; Jahns and Holzwarth, 2012), and in cyanobacteria, the extramembrane phycobiliprotein-containing antennae, the phycobilisomes (PBSs; for review, see Kirilovsky and Kerfeld, 2012; Kirilovsky, 2014). Despite these differences in composition and structure of their antennae, carotenoids have an essential role in both plants and cyanobacteria. In plants, high irradiance leads to acidification of the lumen that triggers conformational changes in the light-harvesting complexes and in their organization in the membrane, switching the light-harvesting complex into an effective energy-dissipating form. In cyanobacteria, high irradiance photoactivates a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), that acts as the stress sensor and the energy quencher. In both cases, changes in pigment-pigment interactions (carotenoid-chlorophyll, carotenoid-bilin, chlorophyll-chlorophyll) enable thermal dissipation of excitation energy via three different possible mechanisms: excitation energy transfer (Ruban et al., 2007; Berera et al., 2013), charge transfer (Holt et al., 2005; Tian et al., 2011), or excitonic interactions between the pigments (Bode et al., 2009).The study of the photoactivation of the OCP and its interaction with the phycobilisome is essential to elucidate the mechanism of energy quenching in cyanobacterial photoprotection. The OCP is a soluble 35-kD protein constituted by an α-helical N-terminal domain (residues1–165) and an α-helix/β-sheet C-terminal domain (residues 190–317; Kerfeld et al., 2003; Wilson et al., 2010; Fig. 1A). A flexible linker of 25 amino acids connects both domains. The ketocarotenoid 3′-hydroxyechinenone (3′-hECN), having a carbonyl (keto) group in one of the rings and a hydroxyl group in the other one, spans both domains of the protein, with the carbonyl group residing in a hydrophobic pocket of the C-terminal domain. Tyr-201 and Trp-288 interact via hydrogen bonds to the carotenoid keto group. In the dark, the OCP is orange (OCP^o). Absorption of blue-green light by the carotenoid induces conformational changes in the carotenoid and in the protein converting the orange form into the active red form (OCP^r; Wilson et al., 2008; Fig. 1C). The photoconversion reaction has a very low quantum yield, and the rate of OCP^r accumulation largely depends on light intensity (Wilson et al., 2008). Thus, accumulation of the red form occurs only under high irradiance (Wilson et al., 2008). Both OCP^o and OCP^r are energetically suitable to quench PBS fluorescence and excitation energy (Polívka et al., 2013; Niedzwiedzki et al., 2014), but only OCP^r is able to bind to the PBS and dissipate most of the excess energy as heat (Gwizdala et al., 2011). In OCP^o, strong interactions exist between the N- and C-terminal globular domains, including salt bridges between residues Trp-277-Asn-104 and Arg-155-Glu-244 (Kerfeld et al., 2003; Wilson et al., 2010). Upon photoactivation, these bonds are broken, leading to the solvent exposure of Arg-155, which plays an essential role in OCP binding to PBS (Wilson et al., 2012; Fig. 1C). The PBSs from Synechocystis sp. PCC 6803 (used in this work and hereafter simply referred to as Synechocystis) are formed by a core of allophycocyanin (APC) trimers. These trimers are organized in three cylinders from which rods containing phycocyanin hexamers radiate (for review, see Grossman et al., 1993; MacColl, 1998; Adir, 2005). OCP^r binds to one APC trimer, and its open structure allows the interaction between the OCP carotenoid and one APC bilin (Wilson et al., 2012). The first site of energy and fluorescence quenching is an APC trimer emitting at 660 nm (Tian et al., 2011, 2012, 2013; Takahashi et al., 2013). Once OCP^r is attached to PBS, thermal dissipation increases and less energy arrives at both photosystems (Wilson et al., 2006; Rakhimberdieva et al., 2010; Gorbunov et al., 2011). When the light becomes less intense, full antenna capacity is required. The Fluorescence Recovery Protein (FRP) is essential for this process. FRP accelerates the OCP^r to OCP^o dark conversion and facilitates OCP detachment from PBS (Boulay et al., 2010; Gwizdala et al., 2011; Sutter et al., 2013). The active FRP is a nonchromophorylated dimer that interacts with the C-terminal domain of the OCP^r (Sutter et al., 2013).Open in a separate windowFigure 1.A and B, Structure of the OCP from Synechocystis sp. PCC 6803 (Protein Data Bank identifier: 3MG1). The OCP monomer is represented in the orange state. The N-terminal arm (residues 1–22; red) interacts with the C-terminal domain (residues 196–315; sky blue). The Pro-22 and the Asp-6 are marked in blue. The N-terminal domain (residues 22–165) is green in the figure, and the linker between N-terminal and C-terminal domains is colored in violet. C, Model of photoactivation. Upon light absorption, the orange OCP^o is converted into the active red OCP^r. Changes in the carotenoid conformation induce conformational changes in the C-terminal domain, leading to the breakage of the interactions between the N-terminal and C-terminal domains and the opening of the protein.Previously, it has been demonstrated that the N-terminal globular domain of the OCP (green in Fig. 1, A and B) is a constitutively active energy quencher (Leverenz et al., 2014). Thus, its interaction with the C-terminal globular domain is essential for inhibiting OCP binding to PBS and energy quenching under low irradiance. This process must be tightly regulated. Little is known about this regulation. One possibility is that the N-terminal arm of the protein (red in Fig. 1, A and B), which in OCP^o interacts with the C-terminal globular domain, could have a role in this regulation.According to the OCP structure Asp-6, could form a hydrogen bond with Arg-229, which could stabilize the closed form of OCP^o. Pro-22 is located at the bent junction between the N-terminal arm and the N-terminal globular domain. It has been proposed that a cis-trans Pro isomerization could be involved in OCP photoactivation (Gorbunov et al., 2011), suggesting that Pro-22 isomerization could help the movement of the N-terminal arm and its detachment from the C-terminal domain during OCP photoactivation. In this work, we studied the effect of deleting the N-terminal arm and the mutations Asp-6-Leu and Pro-22-Val on photoactivity and OCP interaction with PBS and FRP (for the position of the N-terminal arm in the structure of OCP^o, see in Fig. 1, A and B).     

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.     

Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection

The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCP^O, to an active red form, OCP^R. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCP^R. A comparison of the spectroscopic properties of the RCP with OCP^R indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCP^R form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.     

In Vitro Reconstitution of the Cyanobacterial Photoprotective Mechanism Mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803

In conditions of fluctuating light, cyanobacteria thermally dissipate excess absorbed energy at the level of the phycobilisome, the light-collecting antenna. The photoactive Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) have essential roles in this mechanism. Absorption of blue-green light converts the stable orange (inactive) OCP form found in darkness into a metastable red (active) form. Using an in vitro reconstituted system, we studied the interactions between OCP, FRP, and phycobilisomes and demonstrated that they are the only elements required for the photoprotective mechanism. In the process, we developed protocols to overcome the effect of high phosphate concentrations, which are needed to maintain the integrity of phycobilisomes, on the photoactivation of the OCP, and on protein interactions. Our experiments demonstrated that, whereas the dark-orange OCP does not bind to phycobilisomes, the binding of only one red photoactivated OCP to the core of the phycobilisome is sufficient to quench all its fluorescence. This binding, which is light independent, stabilizes the red form of OCP. Addition of FRP accelerated fluorescence recovery in darkness by interacting with the red OCP and destabilizing its binding to the phycobilisome. The presence of phycobilisome rods renders the OCP binding stronger and allows the isolation of quenched OCP-phycobilisome complexes. Using the in vitro system we developed, it will now be possible to elucidate the quenching process and the chemical nature of the quencher.     

The Signaling State of Orange Carotenoid Protein

Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3′-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the β-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.     

Structural Determinants Underlying Photoprotection in the Photoactive Orange Carotenoid Protein of Cyanobacteria

The photoprotective processes of photosynthetic organisms involve the dissipation of excess absorbed light energy as heat. Photoprotection in cyanobacteria is mechanistically distinct from that in plants; it involves the orange carotenoid protein (OCP), a water-soluble protein containing a single carotenoid. The OCP is a new member of the family of blue light-photoactive proteins; blue-green light triggers the OCP-mediated photoprotective response. Here we report structural and functional characterization of the wild type and two mutant forms of the OCP, from the model organism Synechocystis PCC6803. The structural analysis provides high resolution detail of the carotenoid-protein interactions that underlie the optical properties of the OCP, unique among carotenoid-proteins in binding a single pigment per polypeptide chain. Collectively, these data implicate several key amino acids in the function of the OCP and reveal that the photoconversion and photoprotective responses of the OCP to blue-green light can be decoupled.     

Influence of zeaxanthin and echinenone binding on the activity of the Orange Carotenoid Protein

In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here, we tested whether echinenone or zeaxanthin can replace hydroxyechinenone in a study in which the nature of the carotenoid bound to the OCP was genetically changed. In a mutant lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity.     

Essential role of two tyrosines and two tryptophans on the photoprotection activity of the Orange Carotenoid Protein

Photosynthetic organisms have developed photoprotective mechanisms to protect themselves from lethal high light intensities. One of these mechanisms involves the dissipation of excess absorbed light energy into heat. In cyanobacteria, light activation of a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), binding a keto carotenoid, is the key inducer of this mechanism. Blue-green light absorption triggers structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here we report the role in photoconversion and photoprotection of individual conserved tyrosines and tryptophans surrounding the rings of the carotenoid. Our results demonstrate that the interaction between the keto group of the carotenoid and Tyr201 and Trp288 is essential for OCP photoactivity. In addition, these amino acids are responsible for carotenoid affinity and specificity. We have already demonstrated that the aromatic character of Tyr44 and Trp110 interacting with the hydroxyl ring is critical. Here we show that the replacement of Tyr44 by Ser affects the stability of the red form avoiding its accumulation at any temperature, while Trp110Ser is affected in the energy necessary to the orange to red conversion and in the interaction with the antenna. Collectively our data support the idea that the red form is essential for photoprotection but not sufficient. Specific conformational changes occurring in the protein seem to be critical to the events leading to energy dissipation.     

AlphaFold and Structural Mass Spectrometry Enable Interrogations on the Intrinsically Disordered Regions in Cyanobacterial Light-harvesting Complex Phycobilisome

Intrinsically disordered proteins/regions (IDPRs) are a very large and functionally important class of proteins that participate in weak multivalent interactions in protein complexes. They are recalcitrant for interrogations using X-ray crystallography and cryo-EM. The IDPRs observed at the interface of the photosynthetic pigment protein complexes (PPCs) remain much less clear, e.g., the major cyanobacterial light-harvesting complex (PBS) contains an unstructured PB-loop insertion in the phycocyanobilin domain (PB domain) of ApcE (the largest polypeptide in PBS). Here, a joint platform is built to probe such structural domains. This platform is characterized by two-round progressive justifications of in silico models by using the structural mass spectrometry data. First, the AlphaFold-generated 3D structure of the PB domain (containing PB-loop) was justified in the context of PBS. Second, docking the AlphaFold-generated ApcG (a ligand) into the first-step justified structure (a receptor). The final ligand-receptor complex was then subjected to a second-round justification, again, by using unequivocal isotopically-encoded cross-links identified in LC-MS/MS. This work reveals a full-length PB-loop structure modelled in the PBS basal cylinder, free from any spatial conflicts against the other subunits in PBS. The structure of PB domain highlights the close associations of the intrinsically disordered PB-loop with its binding partners in PBS, including ApcG, another IDPR. The PB-loop region involved in the binding of photosystem II (PSII) is also discussed in the context of excitation energy transfer regulation. This work calls attention to the highly disordered, yet interrogatable interface between the light-harvesting antenna complexes and the reaction centers.     

Elucidation of the essential amino acids involved in the binding of the cyanobacterial Orange Carotenoid Protein to the phycobilisome

The Orange Carotenoid Protein (OCP) is a soluble photoactive protein involved in cyanobacterial photoprotection. It is formed by the N-terminal domain (NTD) and C-terminal (CTD) domain, which establish interactions in the orange inactive form and share a ketocarotenoid molecule. Upon exposure to intense blue light, the carotenoid molecule migrates into the NTD and the domains undergo separation. The free NTD can then interact with the phycobilisome (PBS), the extramembrane cyanobacterial antenna, and induces thermal dissipation of excess absorbed excitation energy. The OCP and PBS amino acids involved in their interactions remain undetermined. To identify the OCP amino acids essential for this interaction, we constructed several OCP mutants (23) with modified amino acids located on different NTD surfaces. We demonstrated that only the NTD surface that establishes interactions with the CTD in orange OCP is involved in the binding of OCP to PBS. All amino acids surrounding the carotenoid β1 ring in the OCP^R-NTD (L51, P56, G57, N104, I151, R155, N156) are important for binding OCP to PBS. Additionally, modification of the amino acids influences OCP photoactivation and/or recovery rates, indicating that they are also involved in the translocation of the carotenoid.     

mRNA环结构对蛋白质折叠速率的影响

前期的相关研究发现mRNA二级结构中存在对蛋白质折叠速率的重要影响因素.而mRNA二级结构中普遍存在着各种复杂的环结构,这些环结构是否对蛋白质折叠速率也有重要的影响呢?不同的环结构对蛋白质折叠速率的影响是否相同呢?基于此想法,建立了一个包含mRNA内部环、发夹环、膨胀环和多分支环等环结构信息和相应蛋白质折叠速率的数据库.对于数据库中的每一个蛋白质,计算了mRNA二级结构中各种环结构碱基含量、配对碱基含量及单链碱基含量等参量,分析了各参量与相应蛋白质折叠速率的相关性.结果显示,各种环结构碱基含量与蛋白质折叠速率均呈极显著或显著正相关.说明mRNA环结构对蛋白质折叠速率有重要的影响.进一步,把蛋白质按照不同折叠类型或不同二级结构类型分组后,对每一组蛋白质重复上述的分析工作.结果表明,对不同类蛋白质,mRNA的各种环结构对其相应蛋白质折叠速率的影响存在着显著差异.上述研究将为进一步开展有关mRNA和蛋白质折叠速率的研究奠定理论基础.     

Binding of red form of Orange Carotenoid Protein (OCP) to phycobilisome is not sufficient for quenching

The Orange Carotenoid Protein (OCP) is responsible for photoprotection in many cyanobacteria. Absorption of blue light drives the conversion of the orange, inactive form (OCP^O) to the red, active form (OCP^R). Concomitantly, the N–terminal domain (NTD) and the C–terminal domain (CTD) of OCP separate, which ultimately leads to the formation of a quenched OCP^R–PBS complex. The details of the photoactivation of OCP have been intensely researched. Binding site(s) of OCP^R on the PBS core have also been proposed. However, the post–binding events of the OCP^R–PBS complex remain unclear. Here, we demonstrate that PBS–bound OCP^R is not sufficient as a PBS excitation energy quencher. Using site–directed mutagenesis, we generated a suite of single point mutations at OCP Leucine 51 (L51) of Synechocystis 6803. Steady–state and time–resolved fluorescence analyses demonstrated that all mutant proteins are unable to quench the PBS fluorescence, owing to either failed OCP binding to PBS, or, if bound, an OCP–PBS quenching state failed to form. The SDS–PAGE and Western blot analysis support that the L51A (Alanine) mutant binds to the PBS and therefore belongs to the second category. We hypothesize that upon binding to PBS, OCP^R likely reorganizes and adopts a new conformational state (OCP^3rd) different than either OCP^O or OCP^R to allow energy quenching, depending on the cross–talk between OCP^R and its PBS core–binding counterpart.     

ApcD, ApcF and ApcE are not required for the Orange Carotenoid Protein related phycobilisome fluorescence quenching in the cyanobacterium Synechocystis PCC 6803

In cyanobacteria, strong blue-green light induces a photoprotective mechanism involving an increase of energy thermal dissipation at the level of phycobilisome (PB), the cyanobacterial antenna. This leads to a decrease of the energy arriving to the reaction centers. The photoactive Orange Carotenoid Protein (OCP) has an essential role in this mechanism. The binding of the red photoactivated OCP to the core of the PB triggers energy and PB fluorescence quenching. The core of PBs is constituted of allophycocyanin trimers emitting at 660 or 680nm. ApcD, ApcF and ApcE are the responsible of the 680nm emission. In this work, the role of these terminal emitters in the photoprotective mechanism was studied. Single and double Synechocystis PCC 6803 mutants, in which the apcD or/and apcF genes were absent, were constructed. The Cys190 of ApcE which binds the phycocyanobilin was replaced by a Ser. The mutated ApcE attached an unusual chromophore emitting at 710nm. The activated OCP was able to induce the photoprotective mechanism in all the mutants. Moreover, in vitro reconstitution experiments showed similar amplitude and rates of fluorescence quenching. Our results demonstrated that ApcD, ApcF and ApcE are not required for the OCP-related fluorescence quenching and they strongly suggested that the site of quenching is one of the APC trimers emitting at 660nm. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Carotenoid Changes in the 'Shamouti' Orange Peel During Chloroplast--Chromoplast Transformation on and off the Tree

Seasonal changes in peel carotenoids were investigated in Shamoutioranges on and off the tree. Generally the pattern of changes in carotenoids during the colourtransition of the peel from green to orange (colour break) wassimilar in attached fruit, in fruit detached when still greenand stored under normal conditions at 20°C, and in fruitundergoing de-greening by ethylene. There was a gradual disintegrationof chloroplasts, as shown by the disappearance of chlorophylls.Total carotenoids reached a minimum level and subsequently beganto accumulate. At this point, there was a hypsochromic shiftof 6–8 nm in the absorption spectrum of total carotenoidsindicating not only the completion of the conversion of chloroplaststo chromoplasts but also the appearance of carotenoids of adifferent type. The main changes were the disappearance of ß-carotene,lutein, and neoxanthin, and the appearance of phytofluene, isomersof violaxanthin, various other epoxides and pink apo-carotenals,and methylketone carotenoids. Phytofluene was found to reachrelatively higher concentrations in stored fruit. The observed changes in the composition and amount of carotenoidsappear to characterize the transformation of chloroplasts intochromoplasts in both attached and detached green fruit.     

Phycobilisome: architecture of a light-harvesting supercomplex

The phycobilisome (PBS) is an extra-membrane supramolecular complex composed of many chromophore (bilin)-binding proteins (phycobiliproteins) and linker proteins, which generally are colorless. PBS collects light energy of a wide range of wavelengths, funnels it to the central core, and then transfers it to photosystems. Although phycobiliproteins are evolutionarily related to each other, the binding of different bilin pigments ensures the ability to collect energy over a wide range of wavelengths. Spatial arrangement and functional tuning of the different phycobiliproteins, which are mediated primarily by linker proteins, yield PBS that is efficient and versatile light-harvesting systems. In this review, we discuss the functional and spatial tuning of phycobiliproteins with a focus on linker proteins.     

Phycobilisome Structure of Porphyridium cruentum: POLYPEPTIDE COMPOSITION

Purified phycobilisomes of Porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the α and β subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the γ subunit of phycoerythrin. Another colored polypeptide had a molecular weight of 95,000 and the characteristics of long wavelength-emitting allophycocyanin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin. In addition, the core fraction was enriched in a colored 95,000 dalton polypeptide. Inasmuch as a polypeptide with the same molecular weight is found in thylakoid membranes (free of phycobilisomes), it is suggested that this polypeptide is involved in anchoring phycobilisomes to thylakoid membranes.