CYANOPROTECT: Study of a cyanobacterial photoprotection mechanism involving the Orange Carotenoid protein, a new photoactive carotenoid protein
The ambition of CYANOPROTECT is to create a multidisciplinary network of field-leading, highly -productive laboratories able to unravel the basic molecular mechanism of photoprotection in cyanobacteria, elucidate the photocycle of a new photoactive protein and further understand the role of proteins on the spectral properties of carotenoids.<br />While energy dissipation mechanisms existing in plants and algae have been well-studied, the demonstration of the existence of an energy dissipating mechanism in cyanobacteria is very recent and its molecular mechanism remains to be elucidated. Moreover, this process utilizes a completely different molecular mechanism. The key elements are: the phycobilisome (the cyanobacterial antenna); the OCP, a photoactive carotenoid protein, having a photocycle still unknown, that acts as the inducer of energy dissipation and most probably as the energy quencher; and the FRP, a protein needed for recovey. Our research has as principal objectives the elucidation of 1) the components of the phycobilisome, the cyanobacterial antenna, involved in this photoprotective mechanism; 2) the nature of the energy quencher; 3) the interactions between the different elements involved: OCP, phycobilisomes and FRP. <br />The OCP will be also used as a model protein to study the protein influence on the electronic properties of carotenoid proteins. This is an important task since the carotenoid functions, in photoprotection, in vision and as antioxidant, are tightly bound to their electronic properties. The OCP is an excellent material for this study since it is water-soluble, binds only one carotenoid molecule and many engineered proteins could be easily obtained. <br />Our project could also have an applied interest. The knowledge and strains emerging from this work could help in to determine the better conditions for photoproduction of large cyanobacterial biomass suitable for biotechnological purposes.
The success of the CYANOPROTECT project requires the combination of expertises on biochemistry (isolation of proteins and complexes), genetics (construction of mutants and gene regulation) and physiology (biophysical characterization of photoprotection) of cyanobacteria with expertises in advanced spectroscopy methods allowing the study of dynamic changes of carotenoid and proteins This complementary knowledge is provided by the 2 partners that have already collaborated on various international projects and belong to the same research campus thereby facilitating very frequent discussions. In addition, their international collaboration with field-leading and highly-productive laboratories which are expert on femtosec absorbance kinetics, picoseconds fluorescence kinetics and one single molecule fluorescence measurements (Rink van Grondelle, Amsterdam and Herbert Amerongen, Wageningen), and with crystallographers specialised in the OCP and the cyanobacterial antenna (Cheryl Kerfeld, Berkeley and Noam Adir, Haifa), place them in a very good position to success in this project.
Kirilovsky’s group will construct the cyanobacterial mutants and characterized them while Robert’s group will study the OCP photocycle and the influence of the protein on carotenoid characteristics. R van Grondelle’s group will study the excited states of the carotenoid using ultrafast transient absorption measurements and will try to elucidate the site of energy quenching; that of H van Amerongen will do ultra-rapid fluorescence kinetics measurements to study the energy transfer processes in the “quenched” and “unquenched” phycobilisomes and finally Cheryl Kerfeld and Noam Adir will resolve the structure of FRP and mutated OCPs and of the OCP-APC trimers (or phycobilisomes) complexes (tasks 2 and 3). The OCP mutants and the isolated proteins and complexes needed for the experiments realized in these laboratories will be provided by Kirilovsky’s group.
The results obtained allowed us to propose a new and more complete description of the OCP-photoprotective mechanism. The following are the principal points of the mechanism elucidated in the last two years. Three elements are essential and sufficient for the blue-green light induced photoprotective mechanism, the OCP, the PBs and the FRP. Blue-green light triggers the activation of the OCP inducing conformational changes in the carotenoid and the protein that are needed to attach to the phycobilisome and to quench its fluorescence. Only the activated red protein is able to bind to the phycobilisome. This binding is light independent: once the OCP photoactivated and converted to the red form, it can bind to PBs even in darkness. Only one red OCP is able to quench all the fluorescence of the PB. The OCP binds to the core of the PBs. The primary site of quenching is one of the basal APC trimers emitting at 660nm. This quenching is very fast and efficient and largely decreases the energy arriving to the reaction centers. The OCP N-terminal domain is the active site of the protein. The isolated terminal domain carries the carotenoid and it is able to bind to the PBs and to quench their fluorescence. The Arg155 of the N-terminal domain is essential for this binding. The C-terminal domain is the regulator module of the OCP. The attachment of OCPr to the PBs stabilizes the red activated form that mostly remains attached till FRP will interact with it. The attached OCPr quenches the PB absorbed energy and fluorescence . The FRP by interacting with the attached OCPr induces its conversion to OCPo and its detachment from the phycobilisome. The active FRP is a dimer and Arg60, Asp50 and Trp54 are essential for its activity. The FRP interacts with the C-terminal domain of the OCP. Under strong illumination a new OCPr will rapidly attach to the phycobilisome that will “remain” quenched. In low light fluorescence recovery occurs.
The principal subjects that we will study in the next months are
1) The FRP protein: 1) Characterization of the FRP from Synechocystis; 2) The interaction FRP-OCP by constructing FRP mutants. 3) FRP structure in collaboration with Prof Cheryl Kerfeld
2) The photocycle of OCP using FTIR and ultrafast absorption techniques
3) Influence of OCP mutations on the carotenoid (Raman spectroscopy)
4) Energy dissipation mechanism in collaboration with R van Grondelle’s laboratory
Since the program announced in the ANR project has advanced very quickly new studies will be begun.
1) Characterization of OCPs isolated from other cyanobacteria and study of the specificity of the interaction OCP-phycobilisome. The OCP of Synechocystis is able to induce fluorescence quenching of phycobilisomes isolated from other cyanobacteria strains?
2) Study of the role of genes encoding for the N-terminal part of the OCP.
3) Study of the transcriptional regulation of ocp and frp genes. Study of promoters.
Gwizdala, M, Wilson A, Kirilovsky D (2011) Plant Cell 23 (7) 2631-2643
Lijin Tian, Ivo H.M. van Stokkum, Rob B. M. Koehorst, Aniek Jongerius, Diana L. Kirilovsky, and Herbert van Amerongen (2011) J. Am. Chem. Soc., 133 (45) 18304-18311
D. Kirilovsky, C.A. Kerfeld (2012)Biochim. Biophys. Acta 1817, 158-166
Denis Jallet, Michal Gwizdala and Diana Kirilovsky (2012) Biochim Biophys Acta 1817, 1411-1427
Rudi Berera, Ivo HM van Stokkum, Michal Gwizdala, Adjélé Wilson, Diana Kirilovsky and Rienk van Grondelle (2012) J Physical Chemistry B, 116: 2568-2574
Lijin Tian, Michal Gwizdala, Ivo H.M. van Stokkum, Rob B.M. Koehorst, Diana Kirilovsky and Herbert van Amerongen. (2012) Biophysical Journal, 102 : 1692-1700
Wilson A, Gwizdala M, Mezzetti A, Alexandre M, Kerfeld CA, Kirilovsky D (2012) Plant Cell, Plant Cell, 24 1972-1983
Maria M. Mendes-Pinto, Elodie Sansiaume, Hideki Hashimoto, Andrew A. Pascal, Andrew Gall, and Bruno Robert (2013) J. Phys. Chem. B, 117 11015–11021
Mendes-Pinto, MM, Galzerano, D; Telfer, A; Pascal, AA; Robert, B; Ilioaia, C (2013) J Biol Chem 288, 18758-18765
Kirilovsky D and Kerfeld C (2013) Photochem. Photobiol. Sci., 12: 1135-1143
Sutter M, Wilson A, Leverenz R, Lopez-Igual R, Thurotte A, Salmeen A, Kirilovsky D and Kerfeld C (2013) Proc. Natl. Acad. Sci. USA, 110, 10022-10027
Berera,R, Gwizdala,M, van Stokkum,I, Kirilovsky, D, van Grondelle, Rienk (2013) J Physical Chemistry B 117, 9121-9128
Leverenz, R, Jallet D, Li M-D, Mathies RA, Kirilovsky D, Kerfeld C (2013) Plant Cell (in press)
Jallet D, Thurotte A, Leverenz R, Prreau F, Kerfeld C, Kirilovsky D (2013) Plant Physiol (in press)
The survival and growth of photosynthetic organisms, which renew the Planet oxygenic atmosphere, produce the biomass for the food chain and holds the potential for the production of clean and renewable energy, strongly depends on the optimisation between an efficient collection of light energy to sustain photosynthesis and protection against its photo-oxidising effects. Like plants and algae, cyanobacteria, the most abundant photosynthetic organism on Earth that colonize almost all biotopes from fresh and salt waters to desert soils, decrease the energy arriving from the antenna into the photosynthetic reaction centers by increasing heat dissipation, thereby preventing photo-oxidative damage. While the photoprotection process of plants has been largely studied, its equivalent in cyanobacteria, recently discovered, appeared to use a completely different, but poorly characterized, mechanism.
We have recently shown that in cyanobacteria, the increase of energy dissipation as heat is induced by light activation of a soluble protein containing a single non-covalently bound carotenoid. Absorbance of blue-green light by this Orange-Carotenoid-Protein (OCP) induces reversible structural changes in OCP and its carotenoid, converting the dark stable orange form into a red relatively unstable active form. These results are completely new since OCP is the first photoactive protein containing a carotenoid as the chromophore. Moreover, the OCP photocycle is completely different to those of other photoactive proteins. Almost nothing is known about the OCP photoactivation mechanism and about how the OCP induces photoprotection. Furthermore, we have discovered that another protein, the Fluorescence Recovery Protein (FRP) operates in the photoprotective mechanism. It is essential to recover the fully antenna capacity under low light conditions. The first goal of CYANOPROTECT is to elucidate the molecular mechanisms of OCP and FRP activities to fully understand the related photoprotective process.
In the framework of this project, the OCP will be also used as a model protein to study the protein influence on the electronic properties of carotenoid proteins. This is an important task since the carotenoid functions, in photoprotection, in vision and as antioxidant, are strictly related to their electronic properties. The OCP is an excellent material for this study since it is water-soluble, it binds only one carotenoid molecule and many engineered proteins could be easily obtained in Synechocystis 6803, the principal organism used in this project.
Finally, our CYANOPROTECT project could also have an applied interest. Indeed, the knowledge and the strains emerging from this work could help in the determination of better conditions for the photoproduction of large cyanobacterial biomass suitable for biotechnological purposes.
The success of the CYANOPROTECT project requires the combination of expertises on biochemistry, genetics and physiology of cyanobacteria with expertises in advanced spectroscopy methods allowing the study of dynamic changes of carotenoid and proteins. This complementary knowledge is provided by the 2 partners that have already collaborated on various international projects and belong to the same research campus thereby facilitating very frequent discussions. Both partners are internationally recognized as experts in their fields which publish in high-ranking journals (Nature, PNAS, Plant Cell). In addition, their international collaboration with field-leading and highly-productive laboratories which are expert on femtosecond absorbance kinetics, picosecond fluorescence kinetics and single molecule fluorescence measurements (Rienk van Grondelle, Amsterdam and Herbert van Amerongen, Wageningen), and with crystallographers specialised in the OCP and the cyanobacterial antenna (Cheryl Kerfeld, Berkeley and Noam Adir, Haifa), place them in a very good position to succeed in this project.
Madame Diana Kirilovsky (COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - DIRECTION DU CENTRE DE FONTENAY-AUX-ROSES) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
CEA CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR EST
CEA COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - DIRECTION DU CENTRE DE FONTENAY-AUX-ROSES
Help of the ANR 349,998 euros
Beginning and duration of the scientific project: - 36 Months