The role of far-red spectral states in the energy regulation of phycobilisomes

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.M.G., T.P.J.K. and R.v.G. were supported by R.v.G.'s advanced investigator grant (267333, PHOTPROT) from the European Research Council and TOP grant (700.58.305) from the Foundation of Chemical Sciences part of NWO. T.P.J.K. was additionally supported by the University of Pretoria‘s Research Development Programme (A0W679). R.v.G. gratefully acknowledges his ‘Academy Professor’ grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). M.G. was additionally funded by EMBO, the Claude Leon Foundation and the University of Pretoria.http://www.elsevier.com/locate/bbamem2020-04-01hj2019Physic

Charge transfer states in phycobilisomes

Phycobilisomes (PBs) absorb light and supply downstream photosynthetic processes with excitation energy in many cyanobacteria and algae. In response to a sudden increase in light intensity, excess excitation energy is photoprotectively dissipated in PBs by means of the orange carotenoid protein (OCP)-related mechanism or via a light-activated intrinsic decay channel. Recently, we have identified that both mechanisms are associated with far-red emission states. Here, we investigate the far-red states involved with the light-induced intrinsic mechanism by exploring the energy landscape and electro-optical properties of the pigments in PBs. While Stark spectroscopy showed that the far-red states in PBs exhibit a strong charge-transfer (CT) character at cryogenic temperatures, single molecule spectroscopy revealed that CT states should also be present at room temperature. Owing to the strong environmental sensitivity of CT states, the knowledge gained from this study may contribute to the design of a new generation of fluorescence markers

Single-molecule identification of quenched and unquenched states of LHCII

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.This material is based on work supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Award Number DE-FG02-07ER15892 (to W.E.M) and by the Dutch organization for scientific research (NWO-ALW) via a Vici grant (to R.C.). R.v.G. and T.P.J.K. were supported by the Netherlands Organization for Sciences, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305). R.v.G. was further supported by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT) and by the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his Academy Professor grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). T.P.J.K. was further supported by University of Pretoria’s Research Development Programme (grant no. A0W679). The authors would like to acknowledge the following fellowships: a Postdoctoral Fellowship from the Center for Molecular Analysis and Design at Stanford University (to G.S.S.-C.); a Kenneth and Nina Tai Stanford Graduate Fellowship (to H.-Y.Y.); and a Long Term Fellowship from EMBO (to M.G.).http://pubs.acs.org/journal/jpclcdhb2017Physic

Single-molecule identification of quenched and unquenched states of LHCII

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.This material is based on work supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Award Number DE-FG02-07ER15892 (to W.E.M) and by the Dutch organization for scientific research (NWO-ALW) via a Vici grant (to R.C.). R.v.G. and T.P.J.K. were supported by the Netherlands Organization for Sciences, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305). R.v.G. was further supported by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT) and by the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his Academy Professor grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). T.P.J.K. was further supported by University of Pretoria’s Research Development Programme (grant no. A0W679). The authors would like to acknowledge the following fellowships: a Postdoctoral Fellowship from the Center for Molecular Analysis and Design at Stanford University (to G.S.S.-C.); a Kenneth and Nina Tai Stanford Graduate Fellowship (to H.-Y.Y.); and a Long Term Fellowship from EMBO (to M.G.).http://pubs.acs.org/journal/jpclcdhb2017Physic

Caractérisation in vitro et in vivo du mécanisme de photoprotection lié à l'OCP chez la cyanobactérie Synechocystis PCC6803

Strong light can cause damage and be lethal for photosynthetic organisms. An increase of thermal dissipation of excess absorbed energy at the level of photosynthetic antenna is one of the processes protecting against deleterious effects of light. In cyanobacteria, a soluble photoactive carotenoid binding protein, Orange Carotenoid Protein (OCP) mediates this process. The photoactivated OCP by interacting with the core of phycobilisome (PB; the major photosynthetic antenna of cyanobacteria) triggers the photoprotective mechanism, which decreases the energy arriving at the reaction centres and PSII fluorescence. The excess energy is dissipated as harmless heat. To regain full PB capacity in low light intensities, theFluorescence Recovery Protein (FRP) is required. FRP accelerates the deactivation of OCP.In this work, I present my input in the understanding of the mechanism underlying the OCPrelated photoprotection. I further characterized the FRP of Synechocystis PCC6803, the model organism in our studies. I established that the Synechocystis FRP is shorter than what it was proposed in Cyanobase and it begins at Met26. Our results also revealed the great importance of a high OCP to FRP ratio for existence of photoprotection. The most remarkable achievement of this thesis is the in vitro reconstitution of the OCPrelated mechanism using isolated OCP, PB and FRP. I demonstrated that light is only needed for OCP photoactivation but OCP binding to PB is light independent. Only the photoactivated OCP is able to bind the PB and quench all its fluorescence. Based on our in vitro experiments we proposed a molecular model of OCP-related photoprotection. The in vitro reconstituted system was applied to examine the importance of a conserved salt bridge (Arg155-Glu244) between the two domains of OCP and showed that this salt bridge stabilises the inactive form of OCP. During photoactivation this salt bridge is broken and Arg155 is involved in the interaction between the OCP and the PB. The site of OCP binding in the core of a PB wasalso investigated with the in vitro reconstituted system. Our results demonstrated that the terminal energy emitters of the PB are not needed and that the first site of fluorescence quenching is an APC trimer emitting at 660 nm. Finally, we characterised the properties of excited states of the carotenoid in the photoactivated OCP showing that one of these states presents a very pronounced charge transfer character that likely has a principal role in energy dissipation. Our results strongly suggested that the OCP not only induces thermal energy dissipation but also acts as the energy dissipator.De fortes illuminations peuvent être dommageables voire même létales pour les organismes photosynthétiques. Une des stratégies utilisées pour se protéger de tels effets délétères consiste à augmenter la dissipation thermique de l’énergie absorbée en excès au niveau des antennes. Chez les cyanobactéries une protéine photo-active, l’Orange Carotenoid Protein (OCP), contrôle ce processus. Une fois photo-activée l’OCP interagit avec le coeur des phycobilisomes (PBs, les antennes collectrices majoritaires chez les cyanobactéries) et déclenche le mécanisme, entrainant à la fois une baisse de l’énergie parvenant aux photosystèmes et une diminution de la fluorescence des PBs. L’énergie absorbée en excès est dissipée sous forme de chaleur. Pour que les PBs regagnent leur pleine capacité de transfert, une autre protéine nommée Fluorescence Recovery Protein (FRP) est requise. La FRP accélère la désactivation de l’OCP. Dans ce manuscrit, je vais présenter ma contribution à la compréhension du mécanisme de photo-protection lié à l’OCP.J’ai continué la caractérisation de la FRP chez Synechocystis PCC 6803, organisme modèle utilisé dans nos études. J’ai montré que la FRP de Synechocystis est plus courte que ce qui est indiqué dans Cyanobase, commençant en fait à la méthionine 26. Mes résultats ont aussi révélé que la photo-protection n’a lieu que lorsque le ratio OCP/FRP est élevé.Le plus grand aboutissement de ma thèse a été la reconstitution in vitro du mécanisme de photo-protection lié à l’OCP en utilisant de l’OCP, de la FRP et des PBs isolés. J’ai montré que la lumière est requise uniquement pour la photo-activation de l’OCP et que l’attachement de l’OCP au PB ne demande aucune illumination. Ce n’est qu’une fois photo-activée que l’OCP peut interagir avec le PB et entrainer la diminution de fluorescence (quenching). En se basant sur les résultats obtenus in vitro nous avons proposé un modèle moléculaire pour le mécanisme de photo-protection lié à l’OCP. Le système de reconstitution in vitro a été utilisé pour évaluer l’importance d’un pont salin conservé (Arg155-Glu244) entre les deux domaines de l’OCP et a révélé que celui-ci stabilise la forme inactive de l’OCP. La photo-activation entraine rupture du pont salin, l’Arg155 étant ensuite impliquée dans l’interaction entre OCP et PB. Le site d’attachement de l’OCP au coeur du PB a aussi été étudié en utilisant le système in vitro. Nos résultats ont montré que les émetteurs terminaux du PB ne sont pas requis et que le site primaire de quenching est un trimère d’allophycocyanine émettant à 660nm. Enfin nous avons étudié les propriétés des états excités du caroténoïde dans l’OCP photo-activée, montrant qu’un de ces états a un caractère de transfert de charge très prononcé et peut avoir un rôle principal dans la dissipation de l’énergie. Nos résultats suggèrent fortement que non seulement l’OCP induit dissipation de l’énergie absorbée sous forme de chaleur mais aussi que l’OCP agit directement comme dissipateur d’énergie

Caractérisation in vitro et in vivo du mécanisme de photoprotection lié à l'OCP chez la cyanobactérie Synechocystis PCC6803

De fortes illuminations peuvent être dommageables voire même létales pour les organismes photosynthétiques. Une des stratégies utilisées pour se protéger de tels effets délétères consiste à augmenter la dissipation thermique de l énergie absorbée en excès au niveau des antennes. Chez les cyanobactéries une protéine photo-active, l Orange Carotenoid Protein (OCP), contrôle ce processus. Une fois photo-activée l OCP interagit avec le coeur des phycobilisomes (PBs, les antennes collectrices majoritaires chez les cyanobactéries) et déclenche le mécanisme, entrainant à la fois une baisse de l énergie parvenant aux photosystèmes et une diminution de la fluorescence des PBs. L énergie absorbée en excès est dissipée sous forme de chaleur. Pour que les PBs regagnent leur pleine capacité de transfert, une autre protéine nommée Fluorescence Recovery Protein (FRP) est requise. La FRP accélère la désactivation de l OCP. Dans ce manuscrit, je vais présenter ma contribution à la compréhension du mécanisme de photo-protection lié à l OCP.J ai continué la caractérisation de la FRP chez Synechocystis PCC 6803, organisme modèle utilisé dans nos études. J ai montré que la FRP de Synechocystis est plus courte que ce qui est indiqué dans Cyanobase, commençant en fait à la méthionine 26. Mes résultats ont aussi révélé que la photo-protection n a lieu que lorsque le ratio OCP/FRP est élevé.Le plus grand aboutissement de ma thèse a été la reconstitution in vitro du mécanisme de photo-protection lié à l OCP en utilisant de l OCP, de la FRP et des PBs isolés. J ai montré que la lumière est requise uniquement pour la photo-activation de l OCP et que l attachement de l OCP au PB ne demande aucune illumination. Ce n est qu une fois photo-activée que l OCP peut interagir avec le PB et entrainer la diminution de fluorescence (quenching). En se basant sur les résultats obtenus in vitro nous avons proposé un modèle moléculaire pour le mécanisme de photo-protection lié à l OCP. Le système de reconstitution in vitro a été utilisé pour évaluer l importance d un pont salin conservé (Arg155-Glu244) entre les deux domaines de l OCP et a révélé que celui-ci stabilise la forme inactive de l OCP. La photo-activation entraine rupture du pont salin, l Arg155 étant ensuite impliquée dans l interaction entre OCP et PB. Le site d attachement de l OCP au coeur du PB a aussi été étudié en utilisant le système in vitro. Nos résultats ont montré que les émetteurs terminaux du PB ne sont pas requis et que le site primaire de quenching est un trimère d allophycocyanine émettant à 660nm. Enfin nous avons étudié les propriétés des états excités du caroténoïde dans l OCP photo-activée, montrant qu un de ces états a un caractère de transfert de charge très prononcé et peut avoir un rôle principal dans la dissipation de l énergie. Nos résultats suggèrent fortement que non seulement l OCP induit dissipation de l énergie absorbée sous forme de chaleur mais aussi que l OCP agit directement comme dissipateur d énergie.Strong light can cause damage and be lethal for photosynthetic organisms. An increase of thermal dissipation of excess absorbed energy at the level of photosynthetic antenna is one of the processes protecting against deleterious effects of light. In cyanobacteria, a soluble photoactive carotenoid binding protein, Orange Carotenoid Protein (OCP) mediates this process. The photoactivated OCP by interacting with the core of phycobilisome (PB; the major photosynthetic antenna of cyanobacteria) triggers the photoprotective mechanism, which decreases the energy arriving at the reaction centres and PSII fluorescence. The excess energy is dissipated as harmless heat. To regain full PB capacity in low light intensities, theFluorescence Recovery Protein (FRP) is required. FRP accelerates the deactivation of OCP.In this work, I present my input in the understanding of the mechanism underlying the OCPrelated photoprotection. I further characterized the FRP of Synechocystis PCC6803, the model organism in our studies. I established that the Synechocystis FRP is shorter than what it was proposed in Cyanobase and it begins at Met26. Our results also revealed the great importance of a high OCP to FRP ratio for existence of photoprotection. The most remarkable achievement of this thesis is the in vitro reconstitution of the OCPrelated mechanism using isolated OCP, PB and FRP. I demonstrated that light is only needed for OCP photoactivation but OCP binding to PB is light independent. Only the photoactivated OCP is able to bind the PB and quench all its fluorescence. Based on our in vitro experiments we proposed a molecular model of OCP-related photoprotection. The in vitro reconstituted system was applied to examine the importance of a conserved salt bridge (Arg155-Glu244) between the two domains of OCP and showed that this salt bridge stabilises the inactive form of OCP. During photoactivation this salt bridge is broken and Arg155 is involved in the interaction between the OCP and the PB. The site of OCP binding in the core of a PB wasalso investigated with the in vitro reconstituted system. Our results demonstrated that the terminal energy emitters of the PB are not needed and that the first site of fluorescence quenching is an APC trimer emitting at 660 nm. Finally, we characterised the properties of excited states of the carotenoid in the photoactivated OCP showing that one of these states presents a very pronounced charge transfer character that likely has a principal role in energy dissipation. Our results strongly suggested that the OCP not only induces thermal energy dissipation but also acts as the energy dissipator.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF