- Email: [email protected]
Slow and Fast Fluorescence Quenching of LHCII in Chlamydomonas Reinhardtii Cells
Tuesday, February 14, 2017
Light Energy Harvesting, Trapping, and Transfer 2166-Pos Board B486 Slow and Fast Fluorescence Quenching of LHCII in Chlamydomonas Reinhardtii Cells Roberta Croce, Lijin Tian, Emine Dinc, Laura M. Roy. VU University Amsterdam, Amsterdam, Netherlands. In this work we have studied the process of non-photochemical quenching (NPQ) in vivo in the model green alga C. reinhardtii. NPQ is the process that protects algae and plants from high light damage by dissipating large part of the absorbed energy as heat and which is activated by the low lumenal pH. Despite a large research effort the NPQ mechanism remains debated. The main obstacle to understand NPQ resides in the gap between in vitro and in vivo studies. On the one hand, the complexity of the thylakoid membrane makes it very difficult to obtain molecular information from in vivo experiments. On the other hand, a good in vitro system for the study of the quenching is not available. This has generated to a series of contrasting models that cannot be validated. We have developed a ‘‘minimal NPQ cell’’ which allows us to study the effect of the individual NPQ players in the membrane of living cells. We show that LHCII, the main antenna complexes of algae and plants, exists in different quenching states in the membrane depending on the growth conditions. This difference is suggested to be due to a difference in crowding. However, we also show that LHCII is not able to switch to the quenched conformation in response to pH. Instead, the presence of a very small amount of the protein LHCSR1 is sufficient to induce a large quenching (50%) in a membrane that contains only LHCII when the pH drops to 5.5. The quenching is very fast and completely reversible. Time-resolved fluorescence measurements at room and low temperature show that the quenching occurs in 360 ps and is not induced by additional clustering of LHCII in the membrane. Based on these results we propose a new model that links NPQ to long term acclimation responses. 2167-Pos Board B487 Allophycocyanin from Gracilaria Chilensis and its Recombinant. A Comparative Biophysical and Spectroscopic Study Jorge A. Dagnino-Leone, Marta Bunster, Jose Martinez-Oyanedel, Maria Victoria Hinrichs. Bioquimica y Biologia Molecular, Universidad de Concepcion, Concepcion, Chile. Phycobilisomes (PBS) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of 490680 nm. In Gracilaria chilensis, an eukaryotic red algae, PBS is composed of Phycoerythrin (PE), Phycocyanin (PC) and Allophycocyanin (APC); these proteins possess chromophores which capture energy and then transfer it to photosytems. PBPs are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab)3, which is associated in hexamers (ab)6. Several of this hexamers form cylinder-like structures. PBS has 2 components: antennas and core. The antennas are composed of PE and PC, whose function is to capture energy between 490-570 and 590-625 nm respectively and transfer it to the core. The core is formed by APC, which can absorb energy in the 620-650 nm range. APC emission allows transferring energy to the photosystems with high efficiency. PBS are also composed by linker proteins which allow their correct assembly at the membrane and possibly regulate the energy transfer. APC is also formed by a and b subunits, which possess a phycocyanobilin (PCB) molecule, an open-chain tetrapyrrol molecule covalently attached to a conserved cysteine residue. In the biosynthesis of the chromophore two enzymes are required heme-oxygenase and phycocyanibilin oxidoreductase. The attachment of PCB molecule to APC is mediated by the activity of a residue specific heterodimeric lyase, which is responsible for the final maturation of APC. The objective of the present work is to compare the functional properties of native APC (ApcN) and recombinant APC (ApcR), and to achieve these goal we have used molecular biology, biochemistry and spectroscopic techniques. We purified apcN from Gracilaria chilensis using ammonium sulphate precipitation and ionic exchange, hydroxyapatite and gel filtration chromatography. To express ApcR, we set up a three bi-cistronic vector system, which were cotransformed in E coli and proteins expression were induced with IPTG. ApcR was purified using ammonium sulphate precipitation, ionic exchange, IMAC and gel filtration chromatography. ApcN and ApcR, were verificated by
441a
MALDI-TOF spectrometry and were analyzed by absorption, fluorescence emission and circular dichroism spectra. Our results show that both protein complexes possess equal absorption and fluorescence emission spectra and fluorescence lifetime. The circular dichrosim spectra are basically the same for both proteins. The main difference between these proteins is the thermal stability of ApcR, which has a Tm 5 C degree lower than ApcN. 2168-Pos Board B488 Orange Carotenoid Protein Picosecond Dynamics Changes with Photo and Chemical Activation Yanting Deng1, Mengyang Xu1, Haijun Liu2,3, Robert E. Blankenship2,3, Andrea G. Markelz1. 1 Department of Physics, State University of New York-Buffalo, buffalo, NY, USA, 2Department of Biology, Washington University in St. Louis, St. Louis, MO, USA, 3Department of Chemistry, Washington University in St. Louis, St. Louis, MO, USA. Orange carotenoid protein (OCP) is the only photosensory protein described to date having a carotenoid as the active chromophore. Upon strong greenblue (or white) illumination, OCP goes from the orange inactive state (OCPO) to the red active state (OCPR) [1]. OCPR interacts with the phycobilisome (PB), assisting in thermal energy dissipation. This dissipation diminishes the formation of reactive oxygen which damages the photosynthetic apparatus and leads to cell death [2]. OCP has a large conformational change as it goes from OCPO to OCPR, and this structural change is thought to be essential to the energy quenching interaction with the PB. However, the conversion from inactive to active state has been recently shown to also occur with high chaotrope concentrations. Specifically high concentration NaSCN produces a long lived red state in the absence of photoexcitation [3]. We suggest that both the susceptibility of OCP to large conformational change and its interaction with PB are associated with changes in the long range picosecond structural flexibility. We measure OCP protein flexibility changes with photoactivation and chemical activation using terahertz absorption spectroscopy. Temperature dependent terahertz time domain spectroscopy is measured in the 100 - 290 K range on OCP solutions as a function of illumination and NaSCN concentration. A rapid increase in the THz absorbance is observed in the 180-220 K range. We quantify the picosecond structural flexibility by both the net THz absorption and the dynamical transition temperature, which scales with structural stability. R.E.B acknowledges DOE award DE-FG02-07ER15902 and A.G.M acknowleges NSF awards DBI 1556359 and MCB 1616529, and DOE award DE-SC0016317 for support of the work. 1. Wilson, A. et al. Proc. Natl. Acad. Sci. U. S. A. 105:12075-12080(2008). 2. Wilson, A. et al. Plant Cell, 18:992-1007(2006). 3. King, J.D. et al. FEBS Lett, 588:4561-4565(2014). 2169-Pos Board B489 Light-Harvesting Complex II in Controlled Architectures: Visualizing Changes in Fluorescence Lifetimes Peter G. Adams1, Cvetelin Vasilev2, C. Neil Hunter2, Matthew P. Johnson2. 1 School of Physics and Astronomy, University of Leeds, Leeds, United Kingdom, 2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom. In the first stages of photosynthesis, light-harvesting membrane protein complexes form an interconnected network, absorbing photons and transferring energy as electronic excited states with high efficiency. Light Harvesting Complex II (LHCII) has several important roles, including: (i) acting as an antenna complex for Photosystem II (PSII), (ii) activating NonPhotochemical Quenching (NPQ) and (iii) promoting multi-layer stacking of thylakoid membranes. In this study, we investigated how the optical properties of LHCII depend upon protein-protein and lipid-protein interactions. Microscale array patterns of either single- or multi-layers of LHCII were generated on solid substrates, and studied by atomic force microscopy and fluorescence microscopy with spectral and lifetime imaging. Fluorescence spectra confirmed that the native chromophore organization of LHCII was maintained. Interestingly, LHCII had lower fluorescence lifetimes in multilayers, suggesting that increased LHCII-LHCII interactions promote the quenched state. In other experiments, LHCII was deposited onto solid supports at varying protein density and in the presence or absence of a surrounding lipid membrane. LHCII fluorescence was strongly enhanced when lipids were added, potentially due to decreased self-quenching as the lipids effectively dilute the LHCII membrane concentration, decreasing LHCII-LHCII interactions. These experiments reveal switching between quenched and unquenched states depending on LHCII-LHCII and LHCIIlipid interactions.
Light Energy Harvesting, Trapping, and Transfer 2166-Pos Board B486 Slow and Fast Fluorescence Quenching of LHCII in Chlamydomonas Reinhardtii Cells Roberta Croce, Lijin Tian, Emine Dinc, Laura M. Roy. VU University Amsterdam, Amsterdam, Netherlands. In this work we have studied the process of non-photochemical quenching (NPQ) in vivo in the model green alga C. reinhardtii. NPQ is the process that protects algae and plants from high light damage by dissipating large part of the absorbed energy as heat and which is activated by the low lumenal pH. Despite a large research effort the NPQ mechanism remains debated. The main obstacle to understand NPQ resides in the gap between in vitro and in vivo studies. On the one hand, the complexity of the thylakoid membrane makes it very difficult to obtain molecular information from in vivo experiments. On the other hand, a good in vitro system for the study of the quenching is not available. This has generated to a series of contrasting models that cannot be validated. We have developed a ‘‘minimal NPQ cell’’ which allows us to study the effect of the individual NPQ players in the membrane of living cells. We show that LHCII, the main antenna complexes of algae and plants, exists in different quenching states in the membrane depending on the growth conditions. This difference is suggested to be due to a difference in crowding. However, we also show that LHCII is not able to switch to the quenched conformation in response to pH. Instead, the presence of a very small amount of the protein LHCSR1 is sufficient to induce a large quenching (50%) in a membrane that contains only LHCII when the pH drops to 5.5. The quenching is very fast and completely reversible. Time-resolved fluorescence measurements at room and low temperature show that the quenching occurs in 360 ps and is not induced by additional clustering of LHCII in the membrane. Based on these results we propose a new model that links NPQ to long term acclimation responses. 2167-Pos Board B487 Allophycocyanin from Gracilaria Chilensis and its Recombinant. A Comparative Biophysical and Spectroscopic Study Jorge A. Dagnino-Leone, Marta Bunster, Jose Martinez-Oyanedel, Maria Victoria Hinrichs. Bioquimica y Biologia Molecular, Universidad de Concepcion, Concepcion, Chile. Phycobilisomes (PBS) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of 490680 nm. In Gracilaria chilensis, an eukaryotic red algae, PBS is composed of Phycoerythrin (PE), Phycocyanin (PC) and Allophycocyanin (APC); these proteins possess chromophores which capture energy and then transfer it to photosytems. PBPs are oligomers of a ab heterodimer; it oligomerizes into a trimer (ab)3, which is associated in hexamers (ab)6. Several of this hexamers form cylinder-like structures. PBS has 2 components: antennas and core. The antennas are composed of PE and PC, whose function is to capture energy between 490-570 and 590-625 nm respectively and transfer it to the core. The core is formed by APC, which can absorb energy in the 620-650 nm range. APC emission allows transferring energy to the photosystems with high efficiency. PBS are also composed by linker proteins which allow their correct assembly at the membrane and possibly regulate the energy transfer. APC is also formed by a and b subunits, which possess a phycocyanobilin (PCB) molecule, an open-chain tetrapyrrol molecule covalently attached to a conserved cysteine residue. In the biosynthesis of the chromophore two enzymes are required heme-oxygenase and phycocyanibilin oxidoreductase. The attachment of PCB molecule to APC is mediated by the activity of a residue specific heterodimeric lyase, which is responsible for the final maturation of APC. The objective of the present work is to compare the functional properties of native APC (ApcN) and recombinant APC (ApcR), and to achieve these goal we have used molecular biology, biochemistry and spectroscopic techniques. We purified apcN from Gracilaria chilensis using ammonium sulphate precipitation and ionic exchange, hydroxyapatite and gel filtration chromatography. To express ApcR, we set up a three bi-cistronic vector system, which were cotransformed in E coli and proteins expression were induced with IPTG. ApcR was purified using ammonium sulphate precipitation, ionic exchange, IMAC and gel filtration chromatography. ApcN and ApcR, were verificated by
441a
MALDI-TOF spectrometry and were analyzed by absorption, fluorescence emission and circular dichroism spectra. Our results show that both protein complexes possess equal absorption and fluorescence emission spectra and fluorescence lifetime. The circular dichrosim spectra are basically the same for both proteins. The main difference between these proteins is the thermal stability of ApcR, which has a Tm 5 C degree lower than ApcN. 2168-Pos Board B488 Orange Carotenoid Protein Picosecond Dynamics Changes with Photo and Chemical Activation Yanting Deng1, Mengyang Xu1, Haijun Liu2,3, Robert E. Blankenship2,3, Andrea G. Markelz1. 1 Department of Physics, State University of New York-Buffalo, buffalo, NY, USA, 2Department of Biology, Washington University in St. Louis, St. Louis, MO, USA, 3Department of Chemistry, Washington University in St. Louis, St. Louis, MO, USA. Orange carotenoid protein (OCP) is the only photosensory protein described to date having a carotenoid as the active chromophore. Upon strong greenblue (or white) illumination, OCP goes from the orange inactive state (OCPO) to the red active state (OCPR) [1]. OCPR interacts with the phycobilisome (PB), assisting in thermal energy dissipation. This dissipation diminishes the formation of reactive oxygen which damages the photosynthetic apparatus and leads to cell death [2]. OCP has a large conformational change as it goes from OCPO to OCPR, and this structural change is thought to be essential to the energy quenching interaction with the PB. However, the conversion from inactive to active state has been recently shown to also occur with high chaotrope concentrations. Specifically high concentration NaSCN produces a long lived red state in the absence of photoexcitation [3]. We suggest that both the susceptibility of OCP to large conformational change and its interaction with PB are associated with changes in the long range picosecond structural flexibility. We measure OCP protein flexibility changes with photoactivation and chemical activation using terahertz absorption spectroscopy. Temperature dependent terahertz time domain spectroscopy is measured in the 100 - 290 K range on OCP solutions as a function of illumination and NaSCN concentration. A rapid increase in the THz absorbance is observed in the 180-220 K range. We quantify the picosecond structural flexibility by both the net THz absorption and the dynamical transition temperature, which scales with structural stability. R.E.B acknowledges DOE award DE-FG02-07ER15902 and A.G.M acknowleges NSF awards DBI 1556359 and MCB 1616529, and DOE award DE-SC0016317 for support of the work. 1. Wilson, A. et al. Proc. Natl. Acad. Sci. U. S. A. 105:12075-12080(2008). 2. Wilson, A. et al. Plant Cell, 18:992-1007(2006). 3. King, J.D. et al. FEBS Lett, 588:4561-4565(2014). 2169-Pos Board B489 Light-Harvesting Complex II in Controlled Architectures: Visualizing Changes in Fluorescence Lifetimes Peter G. Adams1, Cvetelin Vasilev2, C. Neil Hunter2, Matthew P. Johnson2. 1 School of Physics and Astronomy, University of Leeds, Leeds, United Kingdom, 2Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom. In the first stages of photosynthesis, light-harvesting membrane protein complexes form an interconnected network, absorbing photons and transferring energy as electronic excited states with high efficiency. Light Harvesting Complex II (LHCII) has several important roles, including: (i) acting as an antenna complex for Photosystem II (PSII), (ii) activating NonPhotochemical Quenching (NPQ) and (iii) promoting multi-layer stacking of thylakoid membranes. In this study, we investigated how the optical properties of LHCII depend upon protein-protein and lipid-protein interactions. Microscale array patterns of either single- or multi-layers of LHCII were generated on solid substrates, and studied by atomic force microscopy and fluorescence microscopy with spectral and lifetime imaging. Fluorescence spectra confirmed that the native chromophore organization of LHCII was maintained. Interestingly, LHCII had lower fluorescence lifetimes in multilayers, suggesting that increased LHCII-LHCII interactions promote the quenched state. In other experiments, LHCII was deposited onto solid supports at varying protein density and in the presence or absence of a surrounding lipid membrane. LHCII fluorescence was strongly enhanced when lipids were added, potentially due to decreased self-quenching as the lipids effectively dilute the LHCII membrane concentration, decreasing LHCII-LHCII interactions. These experiments reveal switching between quenched and unquenched states depending on LHCII-LHCII and LHCIIlipid interactions.