No description available.

The goal of this research is to investigate the role of the slr1098 gene in the cyanobacterium Synechocystis sp. PCC 6803, a gene with similarity to cpcE which encodes a subunit of an enzyme involved in bilin attachment to phycocyanin. This protein is hypothesized to be involved in oligomerization of phycocyanin due to previous results showing the mutant made shorter phycocyanin rods. The recombinant Slr1098 protein was produced and purified from E. coli cells. Binding assays showed interaction between Slr1098 and both apo- and holo-phycocyanin, but not to apo-allophycocyanin. Slr1098 blocked bilin addition at Cys-82 on CpcB by the CpcS/CpcU bilin lyase. Size exclusion chromatography and sucrose density gradient analysis of complexes formed suggest that Slr1098 strongly interacts with all intermediate forms of phycocyanin and may be an important checkpoint in the biosynthesis and oligomerization of this protein, but that by itself, Slr1098 does not increase oligomerization of phycocyanin.

Phycoerythrin (PE) present on the outer phycobilisome (PBS) rods in Fremyella diplosiphon contains covalently attached phycoerythrobilin (PEB) chromophores for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions. The cpeY and cpeZ genes in F. diplosiphon were shown to attach PEB on alph-82 of PE. To better understand the individual functions of cpeY and cpeZ in native cyanobacteria, we characterized PBS and PE purified from cpeY and cpeZ deletion mutants and compared them with wild type (WT). Both cpeY and cpeZ mutants generated much less PE than WT as well as assembling much less PE into the PBS. PE purified from cpeY mutant had phycocyanobilin on alpha-PE in place of PEB. The mutation of cpeZ affected the biosynthesis and accumulation of beta-PE with a red-shifted absorbance compared to WT PE. CpeY was shown to function as a bilin lyase, and CpeZ possibly functions as a chaperone.

No description available.

Les cyanobactéries sont des organismes procaryotes photosynthétiques. Si l’énergie leur est essentielle, elle peut également être délétère. Afin de se protéger, elles ont acquis plusieurs mécanismes de photoprotection. La thématique de ma thèse est l’étude de l’un d’entre eux par des approches combinées de biologie moléculaire, biochimie et biophysique.Les antennes collectrices de lumière des cyanobactéries sont des complexes extra-membranaires solubles appelés les phycobilisomes. Ils permettent de canaliser l'énergie vers les centres réactionnels des photosystèmes. Sous forte lumière, l'afflux d’énergie y parvenant crée notamment des espèces réactives de l’oxygène, ce qui est délétère pour la cellule. L’Orange Carotenoid Protein (OCP) est impliquée dans un mécanisme de photoprotection qui diminue l'énergie arrivant au niveau des centres réactionnels en augmentant la part d’énergie dissipée sous forme de chaleur. L’OCP est une caroténo-protéine composée de deux domaines globulaires N- et C-terminal qui lie un caroténoïde. Cette protéine photoactivable est à la fois le senseur, et l’acteur du mécanisme de photoprotection. Le mécanisme est désactivé par une seconde protéine, la FRP (Fluorescence Recovery Protein).Le premier chapitre de ce travail de thèse rapporte l’étude de la spécificité des OCPs isolées chez deux souches différentes pour différentes classes de phycobilisomes dont l’architecture du cœur diffère. Le second chapitre présente la méthode mise au point au laboratoire de production de l’OCP chez E.coli, ainsi que la caractérisation d’OCPs clonées depuis le génome de Synechocystis, A. variabilis et A. platensis et surexprimés chez E. coli. Le troisième présente la structure tridimensionnelle du domaine N-terminal, qui est le domaine effecteur de l’OCP. Dans ce chapitre, nous démontrons que le cofacteur caroténoïde se déplace de 12 angstrom au sein de l’OCP lors de la photoactivation. Le quatrième rapporte que le bras N-terminal de l’OCP est une structure singulière qui maintient la protéine fermée à l’obscurité, évitant que l’OCP ne s’active sous faible lumière, ou à l’obscurité. Le cinquième présente la résolution de la structure et l’identification du site actif de la FRP qui nous ont permis de prédire in silico le site d’attachement putatif de la FRP sur le domaine C-terminal de l’OCP. Dans le chapitre 6, je rapporte que deux résidus, l’aspartate 220 et la phénylalanine 299, sont requis pour que l’activité de la FRP soit maximale, confirmant le site d’interaction prédit. / Cyanobacteria, a photosynthetic prokaryote organism, harvest light for living. But harvesting too much light can be harmful. To protect themselves against this stress, cyanobacteria have developed several photoprotective mechanisms. This manuscript reports my work about one of them by combined technics of molecular biology, biochemistry and biophysics.Cyanobacterial light harvesting antennae are extra-membranous complexes called phycobilisomes. They funnel harvested energy into the photosynthetic reaction centers. Under high light, high energy input induces the formation of reactive oxygen species (ROS), which are harmful in excess. One of the existent photoprotective mechanisms helps to avoid ROS formation by decreasing the energy arriving at the reaction centers. The main actor of this mechanism is the photoactive Orange Carotenoid Protein (OCP) that binds to the phycobilisome, and induces an increase of the part of energy dissipated as heat. The OCP is a protein composed by two globular domains (called N- and C- terminal) and binds a carotenoid cofactor. High intensity of blue-green light triggers conformational changes in the inactive orange OCP, which turns red and is now able to binds the PBs. Under low light conditions, this mechanism is turned off by another protein, the Fluorescence Recovery Protein (FRP).The first chapter of this manuscript reports the study of the specificity of OCPs isolated from two strains for different classes of phycobilisomes with different core architecture. The second describe the development of a method to produce holoOCP in E. coli cells. Furthermore, it reports the characterization of the Synechocystis, A. variabilis and A. platensis OCPs isolated from E. coli. The third chapter presents the tridimensional structure of the active N-terminal domain of the OCP. In this chapter, we demonstrate that the carotenoid undergoes a 12anstrom movement upon photoactivation. The fourth chapter rapports that the N-terminal arm of the OCP helps to maintain closed the inactive orange OCP in darkness or low light, avoiding OCP activation and consequent unwanted PBs fluorescence quenching. The fifth presents the resolution of the structure and the identification of the active site of the FRP. These data allow to compute a predictionnal model of interaction between OCP and FRP. I assessed the validity of the model by isolating several modified OCPs. Results shown in chapter 6 report that the aspartate 220 end the phenylalanine 299 are required for effective FRP action.

La lumière est essentielle pour les organismes photosynthétiques qui convertissent l'énergie solaire en énergie chimique. Cependant, la lumière devient dangereuse lorsque l'énergie qui arrive aux centres réactionnels de l'appareil photosynthétique, est en excès par rapport à l’énergie consommée. Dans ce cas, la chaîne de transport d'électrons photosynthétiques se réduit et les espèces réactives de l'oxygène (ROS) sont accumulées, notamment au niveau des deux photosystèmes, PSI et PSII. Les cyanobactéries ont développé des mécanismes photoprotecteurs qui diminuent l'énergie transférée au PSII atténuant ainsi l'accumulation de ROS et les dommages cellulaires, comme l’extinction non-photochimique (NPQcya) induite par la lumière bleue-verte. La soluble Orange Caroténoïde Protéine (OCPo) est essentielle pour ce mécanisme de photoprotection. L'OCP agit comme un senseur de l’intensité lumineuse et un inducteur de la dissipation d'énergie des phycobilisomes (PBS), l'antenne extra-membranaire des cyanobactéries. L'OCP est la première protéine photo-active à caroténoïde connue comme senseur. Une forte lumière bleue-verte déclenche des changements structurels dans l'OCPo qui induisent une forme active, rouge (OCPr). Le domaine N-terminal de l’OCPr, en s’intercalant entre les trimères externes d’un des cylindres basaux du cœur du PBS, augmente la dissipation thermique de l'énergie au niveau de l'antenne. L'OCP possède aussi une autre fonction : l’extinction de l’oxygène singulet, qui protège les cellules du stress oxydatif. Pour récupérer pleinement la capacité de l’antenne en faible lumière, une deuxième protéine est nécessaire, la "Fluorescence Recovery Protein" (FRP), dont le rôle est de détacher l’OCPr des PBS et d’accélérer sa reconversion en OCPo inactive. Ce manuscrit est un état des lieux des connaissances et des dernières avancées sur le mécanisme de NPQ associé à l'OCP dans les cyanobactéries. / Photosynthetic organisms use light energy from the sun in order to perform photosynthesis and to convert solar energy into chemical energy. Absorbance of excess light energy beyond what can be consumed in photosynthesis is dangerous for these organisms. Reactive oxygen species (ROS) are formed at the reaction centers and collecting light antennas inducing photooxidative damage which can lead to cell death. In cyanobacteria, one of these photoprotective mechanisms consists to reduce the amount of energy arriving to the reaction centers by thermal dissipation of the excess absorbed energy. Energy dissipation is accompanied by a decrease of Photosystem II-related fluorescence emission called non-photochemical quenching (NPQ). The soluble Orange Carotenoid Protein (OCPo) is essential for this photoprotective mechanism. The OCP is the first photo-active protein with a carotenoid known as light intensity sensor and acts as energy quencher of the phycobilisome (PB), the extra-membrane antenna of cyanobacteria. Structural changes occur when the OCPo absorbs a strong blue-green light leading to a red active form (OCPr). The N-terminal domain of OCPr burrows into the two external trimers of the core basal APC cylinders of the PB and increases thermal energy dissipation at the level of antenna. The OCP has an additional function in photoprotection as oxygen singlet quencher protecting cells from oxidative stress. Under low light conditions, to recover the full antenna capacity, a second protein is needed, the "Fluorescence Recovery Protein" (FRP), whose role is to detach the OCPr from the PB and accelerate its conversion into an inactive OCPo. In this manuscript, I will review the knowledge about the OCP, since the discovery of the mechanism and its characterization to the latest advances on the OCP-related-NPQ mechanism in cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that able to produce oxygen. They have light harvesting complexes called phycobilisomes (PBS). PBS are generally composed of an allophycocyanin core with phycocyanin and phycoerythrin rods connected to the core. PBS are able to efficiently harvest light energy from different wavelengths of visible light due to the evolution of PBP. Phycoerythrin has five chromophores that are attached to six cysteine residues and is essential for efficient green light capture and transfer of energy for use in photosynthesis. The attachment of these chromophores to PBP is facilitated by enzymes known as bilin lyases. In this study, we characterize and explore the role of enzymes that are involved in the biosynthesis of phycoerythrin in cyanobacteria. Biochemical and molecular techniques were used in the characterization of these proteins to gain a better understanding of their roles in the post-translational modification of phycobiliprotein. In F. diplosipohon, the lyase activity of CpeT was characterized and studied using a heterologous, co-expression system in E. coli. It was determined that CpeT was able to ligate PEB to Cys-165 of CpeB in the presence of CpeZ, a chaperone-like protein. Next, the roles of three proteins, MpeY from RS9916 and MpeQ and MpeW from A15-62, were analyzed using a combination of gene-interruption mutants and recombinant protein expression techniques. The absence of mpeY resulted in the reduction of PEB chromophorylation of MpeA in green light conditions, and recombinant protein coexpression confirmed that MpeY was responsible for PEB attachment to Cys-83 of MpeA. The interruption of mpeQ in A15-62 resulted in a reduced PUB phenotype in MpeA in blue light. Recombinant protein expressions revealed that MpeQ was a lyase-isomerase responsible for the attachment of PUB to Cys-83 of MpeA. Two regulatory proteins located in two conserved configurations of a genomic island present in species that are able to change their phycobilin content in response to different light environments, known as Type-IV chromatic acclimation (CA4), were investigated. FciA and FciB from RS9916 were studied using gene interruption mutants from RS9916 and they were found to be responsible for the CA4 response in CA4-A containing species of Synechococcus.

Cyanobacteria are photosynthetic organisms that efficiently capture light by utilizing the light-harvesting complexes called phycobilisomes. In many cyanobacteria, phycobilisomes are composed of an allophycocyanin core with phycocyanin and phycoerythrin (PE) rods radiating from the core. These phycobiliproteins have multiple bilin chromophores, such as phycoerythrobilin (PEB), covalently attached to specific cysteine (Cys) residues for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions. This study mainly focuses on characterizing the roles of enzymes that are involved in the biosynthetic pathway of the phycobiliproteins within two cyanobacteria Thermosynechococcus elongatus and Fremyella diplosiphon. A combination of molecular and biochemical techniques were used to better understand the roles of these proteins in the post-translational modification and/or stability of phycobiliproteins. Using a heterologous plasmid coexpression system in E. coli, recombinant CpcS-III from T. elongatus was shown to ligate three different bilins to both subunits of allophycocyanin and to the beta subunit of phycocyanin, thus, acting as a bilin lyase. The crystal structure of CpcS-III was also solved, the first bilin lyase structure. Next, the roles of three proteins from F. diplosiphon CpeY, CpeZ, and CpeF were analyzed using a combination of gene knock-out mutants and recombinant protein expression techniques. In the absence of cpeY, chromophorylation to the alpha subunit of PE at Cys-82 was reduced, coinciding with the recombinant data that CpeY is the lyase that attaches PEB to this site. Removing cpeZ from the genome resulted in the destabilization and reduced accumulation of PE, especially the beta subunit CpeB. Recombinant CpeZ was shown to act like a chaperone-like protein and increased the solubility and fluorescence of both recombinant and native CpeB by increasing the stability of the phycobiliprotein and/or by increasing the activities of other lyases. The deletion of cpeF resulted in a reduced-PE phenotype with the doubly attached PEB missing from CpeB at Cys-48/Cys-59. Recombinant CpeF was shown to ligate PEB to CpeB-Cys-48/Cys-59 in the presence of recombinant CpeS (lyase attaches PEB to CpeB-Cys-80) and CpeZ. CpeF also showed a chaperone-like function by stabilizing CpeB, but its main role appears to be as a bilin lyase.

Die meisten Cyanobakterien und Chloroplasten von Rotalgen verfügen über Phycobilisomen, große lichtsammelnde Multiproteinkomplexe, die an der cytoplasmatischen Seite der Thylakoidmembran gebunden vorliegen. Unter stickstofflimitierten Bedingungen werden die Phycobilisomen proteolytisch abgebaut. Dieser Prozess schützt vor Fotoschäden unter den gegebenen Stressbedingungen und liefert gleichzeitig einen großen Vorrat an Stickstoff enthaltenden Substanzen. Das Gen nblA, welches in allen Phycobilisomen enthaltenden Organismen vorkommt, kodiert für ein Polypeptid von ungefähr 7 kDa, das eine Schlüsselrolle im Abbau der Phycobilisomen einnimmt. Die Wirkungsweise von NblA dabei wird jedoch bisher kaum verstanden. Ein Selenomethioninderivat von NblA des filamentösen Cyanobakteriums Anabaena sp. PCC 7120 wurde in Escherichia coli rekombinant hergestellt, gereinigt und kristallisiert. Die Röntgenkristallstruktur von NblA wurde mithilfe der single-wavelength anomalous dispersion-Methode bis zu einer Auflösung von 1,8 Angstrom bestimmt. Das finale Modell verfügt über einen kristallographischen R-Wert von 18,2% und einen freien R-Wert von 21,7%. Das kleine NblA-Protein von 65 Aminosäuren besteht aus zwei alpha-Helices, die in einem ca. 37°-Winkel in einer antiparallelen, V-förmigen Anordnung zueinander stehen. Zwei dieser Monomere bilden die grundlegende strukturelle Einheit von NblA, ein vier-Helix-Bündel, bei dem sich die Spitzen der "Vs" auf der gleichen Seite des Dimers überlagern und über eine nicht-kristallographische zweizählige Achse miteinander verknüpft sind. Auf der Grundlage von Bindungsstudien und der Kenntnis der NblA-Struktur konnte ein Modell für die Bindung von NblA an die Phycobilisomenstruktur postuliert werden. / Cyanobacterial light harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. To gain deeper insights into the mode of action of NblA in this degradation process the crystal structure of NblA was determined and a model of its binding to phycobilisomes was proposed. For this purpose, NblA from Anabaena sp. PCC 7120 was produced as selenomethionine NblA derivative in Escherichia coli B834 (DE3), purified and crystalized. NblA crystals grew as long, but thin rods and belong to the monoclinic space group P2(1) with cell parameters of a = 43.2 A, b = 95.9 A, c = 104.8 A and Beta = 97.0°. They contain twelve NblA monomers in the asymmetric unit. The crystal structure with a resolution of 1.8 A was determined using the single-wavelength anomalous dispersion (SAD) technique and refined to final values for Rwork and Rfree of 0.182 and 0.217, respectively. The small NblA polypeptide of 65 amino acids consists of two alpha-helices which are assembled at a ~37° angle in an antiparallel, V-shaped arrangement. Two NblA monomers form the basic structural unit of NblA, a four-helix bundle with the tips of the V superimposing on the same side of a dimer. The dimer is formed by two molecules related by a non-crystallographic dyad axis. Based on the crystal structure presented here, pull-down experiments, and peptide scan data a model of binding of NblA to phycobilisomes was proposed. Considering the entire phycobilisome structure, NblA is predicted to bind via its amino acids Leu51 and Lys53 to the trimer-trimer interface of the phycobiliprotein hexamers.