Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photoch... more Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photochemical quenching for photoprotection of plants under highlight conditions. The activation mechanism of PsbS and its pH-induced conformational changes are currently unknown. For structural investigation of PsbS, effective synthesis of PsbS with selective isotope or electron-spin labels or non-natural amino acids incorporated would be a great asset. This communication presents cell-free expression as a successful method for in vitro production of PsbS that would allow such incorporation. We have optimized the cell-free method to yield soluble PsbS of ~500 ng/µl using a continuous-exchange method at 30 0 C, along with a successful purification and refolding of PsbS in n-Dodecyl β-D-maltoside (β-DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.
behavior can be different within two or three cell size distance because of the chemical gradient... more behavior can be different within two or three cell size distance because of the chemical gradient in that distance. Therefore, in the modeling of such systems, a spatial resolution is required to be comparable to the cell size. Such spatial resolution poses an extra challenge in the development and execution of the computational model due to the agents sitting over multiple control volumes. In this presentation, a new computational method for cell surface-based reaction for the finite volume method will be introduced and compared to other methods in terms of accuracy and computation speed.
We investigated the temperature-mediated reassociation of the B820 subunit of Rs. rubrum to form ... more We investigated the temperature-mediated reassociation of the B820 subunit of Rs. rubrum to form a light-harvesting 1 complex (LH 1). By combining several spectroscopic techniques with global spectral data analysis fitting, we present evidence for the occurence of two spectral intermediates that appear during the reassociation process. At high temperatures, halfway the reassociation reaction, a prominent intermediate appears that has an absorption maximum around 850 nm, a fluorescence maximum around 860-867 nm, a high anisotropy (0.3 to 0.4) and a circular dichroism spectrum with three or four bands with alternating signs. At lower temperatures, more towards the end of the reassociation process, a second intermediate tends to appear that has an absorption maximum around 860 nm, a fluorescence maximum around 885 nm, a medium to high anisotropy (0.1 to 0.3) and a circular dichroism spectrum with two bands with alternating signs. The latter circular dichroism spectrum has a blueshifted...
Background: The peripheral light-harvesting complexes of photosystem II (LHCII) switch between li... more Background: The peripheral light-harvesting complexes of photosystem II (LHCII) switch between light energy capturing and dissipative states. Results: An NMR analysis is presented of the arginines in LHCII prepared in its two states. Conclusion: The protein core of arginine-glutamate interlocked helices is preserved, whereas moderate changes occur in the stromal loop region. Significance: This work reveals consequences for the molecular switching of LHCII. Light-harvesting antennae of the LHC family form transmembrane three-helix bundles of which two helices are interlocked by conserved arginine-glutamate (Arg-Glu) ion pairs that form ligation sites for chlorophylls. The antenna proteins of photosystem II have an intriguing dual function. In excess light, they can switch their conformation from a light-harvesting into a photoprotective state, in which the excess and harmful excitation energies are safely dissipated as heat. Here we applied magic angle spinning NMR and selective Arg isotope enrichment as a noninvasive method to analyze the Arg structures of the major light-harvesting complex II (LHCII). The conformations of the Arg residues that interlock helix A and B appear to be preserved in the light-harvesting and photoprotective state. Several Arg residues have very downfield-shifted proton NMR responses, indicating that they stabilize the complex by strong hydrogen bonds. For the Arg C ␣ chemical shifts, differences are observed between LHCII in the active, light-harvesting and in the photoprotective, quenched state. These differences are attributed to a conformational change of the Arg residue in the stromal loop region. We conclude that the interlocked helices of LHCII form a rigid core. Consequently, the LHCII conformational switch does not involve changes in A/B helix tilting but likely involves rearrangements of the loops and helical segments close to the stromal and lumenal ends. The elementary step in photosynthesis is the capture of solar energy by the light-harvesting antenna. In eukaryotic organisms, this step is performed by a family of pigment-binding proteins called light-harvesting complexes (LHCs) 2 that absorb sunlight and transfer the excitation energy toward the reaction centers, where charge separation takes place (1). The Lhcb antenna proteins of higher plants and moss and Lhcbm antenna proteins of LHCs in green algae contain three membrane-spanning helices A, B, and C, and their tertiary structure is typified by two crossing A/B helices interconnected by Arg-Glu ion pairs that form ligation sites for Chls. The most abundant LHC in plants and green algae is the major light-harvesting complex II (LHCII), which captures about 50% of all land-bound chlorophylls. The LHCII complex is trimeric, and each monomer forms a scaffold for eight Chla and six Chlb molecules, two luteins (Lut), one neoxanthin, and one violaxanthin, which is reversibly replaced by zeaxanthin upon de-epoxidation during the xanthophyll cycle (2-4). In addition to their light-harvesting function, LHCII complexes are involved in several regulatory mechanisms that balance the incoming excitation energies and prevent photodamage (1, 4-6). Under high sunlight conditions, the photosynthetic antenna can rapidly switch from light-harvesting into a photoprotective state in which excess light energy is safely dissipated as heat (7). This photoprotective mechanism is called non-photochemical quenching (NPQ) and protects oxygenic organisms against photooxidative damage. The major component of NPQ, qE, depends on the transmembrane proton gradient ⌬pH. In plants, a decrease in lumenal pH triggers protonation of PsbS (8, 9) and of LHCs in photosystem II (10) and activates the conversion of the LHC-bound xanthophyll cycle carotenoid from violaxanthin to zeaxanthin (11). In green algae, PsbS is absent, and the NPQ state is triggered by LhcSR, a pigment-binding complex with a short fluorescence lifetime that senses the decrease in lumenal pH (12-14). In algae, LHCII also participates in quenching, in particular the Lhcbm1 com
We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum ... more We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum rubrum from the separated R BChl 2 subunits (B820) and the oligomerization of the B820 subunit from its monomeric peptides. The full LH1 complex was reversibly associated from B820 subunits by either varying the temperature in the range 277-300 K or by varying the detergent concentration in the buffer from 0.36 to 0.52% n-octyl-D-glucopyranoside. Temperature-induced transition measurements showed hysteresis: raising the temperature induced dissociation of B873 directly into B820 subunits whereas upon recooling an intermediate spectral form was observed with an absorption maximum located around 850 nm. This intermediate form was also observed in detergent-induced transitions. It is speculated that the B850 form is a small aggregate of B820, for instance a dimer. Additionally, during a temperaturemediated transition at low detergent concentration, a set of spectral forms with maxima slightly blueshifted from 873 nm were observed, possibly due to opened rings with one or only a few R BChl 2 units missing. The temperature-induced transition of LH1 is discussed in terms of a simple assembly model. It is concluded that a moderately cooperative assembly explains the formation of small aggregates of B820 as well as of incomplete rings. Furthermore, the B820 subunits were reversibly dissociated into the monomeric B777 form by increasing either the temperature or the detergent concentration. Estimations of the enthalpy and entropy changes for the dimeric association reaction of B777 into B820 yielded an enthalpy change of-216 kJ mol-1 and an entropy change of-0.59 kJ mol-1 K-1 , at a detergent concentration of 0.8% n-octyl-D-glucopyranoside.
For the first time, data are presented on the time-resolved disassembly reaction of a highly orga... more For the first time, data are presented on the time-resolved disassembly reaction of a highly organized membrane protein complex in vitro. The photosynthetic core light-harvesting complex of the bacterial strain Rhodospirillum rubrum G9 consists of 12-16 dimeric subunits that in vivo are associated with the photosynthetic reaction center in a ringlike manner. Isolated in a detergent solution, its appearance either as a ringlike complex (called B873 and absorbing at 873 nm), subunit dimer (called B820 and absorbing at 820 nm), or monomeric form (called B777 and absorbing at 777 nm) is strongly temperaturedependent. In thermodynamic equilibria between B820 and B873, intermediate-sized complexes have also been observed that have absorption maxima around 850 nm. It is unknown whether these structures appear as intermediates in the kinetic B820-B873 (dis)assembly reaction. In this paper disassembly of the light-harvesting complex into its dimeric subunits was followed spectroscopically on a time scale up to 200 ms, upon applying an infrared laser-induced temperature jump. The full dissociation process appears to take place on a time scale of tens to hundreds of milliseconds, the rates becoming faster at higher starting temperatures. Applying the same technique, the dissociation reaction of dimeric subunits into monomers also could be established. This dissociation process occurred on a much faster time scale and took place within the 500 µs response time of our detection system.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011
The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and... more The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in 13 C-13 C dipolar homonuclear correlation spectra on a uniformly 13 C-labeled sample. In particular, we were able to resolve several chlorophyll 13 1 carbon resonances that are sensitive to hydrogen bonding to the 13 1-keto carbonyl group. The data show that 13 C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in lightharvesting regulation through MAS solid-state NMR.
In this work polarized picosecond fluorescence measurements were performed on isolated tryptophan... more In this work polarized picosecond fluorescence measurements were performed on isolated tryptophan and tryptophan in a small 22-mer peptide using a streak camera coupled to a spectrograph as a detection system. In both cases the fluorescence decay was multiexponential with decay times of ∼500 ps and ∼4 ns. Surprisingly, also a short-lived (∼16 ps) isotropic fluorescence component was found for both tryptophan and the peptide which, to our knowledge, has never been observed before. Fluorescence anisotropy data showed rotational correlation times of 155 ps and 1.5 ns for the peptide, reflecting local and overall peptide dynamics, respectively. Recently it was argued [Lakowicz, J. R. Photochem. Photobiol. 2000, 72, 421] that the nonexponential fluorescence decay of tryptophan in proteins is mainly due to spectral relaxation, whereas for isolated tryptophan it is due to different rotamers. Our results do not support this view. In contrast we conclude in both cases that the different fluorescence decay times should be ascribed to different rotameric states.
Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowe... more Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting fa...
In plant chloroplast membranes, non-photochemical quenching (NPQ) is activated as a response to a... more In plant chloroplast membranes, non-photochemical quenching (NPQ) is activated as a response to a low luminal pH and controlled by the pH-sensing protein PsbS. It has been proposed that PsbS directly interacts with the light-harvesting complexes (LHCII) of Photosystem II, inducing quenching of LHCII Chl excitations, whilst others proposed that PsbS has an indirect role in controlling the organization of the membrane. In this study, we systematically test the influence of low pH and PsbS on the fluorescence lifetimes of membrane-embedded spinach LHCII. The proteoliposome preparations contain LHCII in mild quenched states, aimed to mimic fluorescence conditions of dark-adapted leaves. We find that under those conditions, acidification and the presence of PsbS do not have significant effect on the LHCII Chl fluorescence lifetimes. This supports a view in which the functional role of PsbS consists of re-organizing the thylakoid membrane under light stress, rather than creating direct qu...
Photosynthetic light-harvesting antenna complexes (LHCs) of plants, moss and green algae form dyn... more Photosynthetic light-harvesting antenna complexes (LHCs) of plants, moss and green algae form dynamic switches between light harvesting and excitation-quenched, dissipative states. This mechanism protects the photosynthetic apparatus under light stress via a photo protective membrane response. Herein, we demonstrate the application of solid-state NMR spectroscopy to wild type, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and purified Cr Light harvesting Complex II (LHCII) reconstituted in thylakoid lipid membranes, to detect the conformational dynamics of LHCII under native conditions. We find that membrane-reconstituted LHCII contains sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Furthermore, plasticity is observed in the N-terminal stretch and in the trans-membrane helical edges facing the thylakoid lumen. In intact thylakoids, the dynamics of these protein and pigment sites is significantly reduced. We con...
The membrane protein Photosystem II subunit S (PsbS) is a pH sensor that plays an essential role ... more The membrane protein Photosystem II subunit S (PsbS) is a pH sensor that plays an essential role in signaling light stress in plants to prevent photo oxidation and generation of detrimental reactive species. PsbS detects thylakoid lumen acidification in excess light conditions via two glutamates facing the lumen, however, its molecular mechanism for activation is elusive. We performed a spectroscopic analysis of wild type Physcomitrella patens PsbS and of mutants in which the active glutamates have been replaced (E69Q, E174Q and E69Q / E174Q). We discovered that E69 exerts allosteric control of PsbS dimerization, while E174 is essential for the secondary structural response to low pH. Based on our results, we propose a molecular pH response mechanism that involves a change in the amphiphatic short helix containing E174 facing the lumen, moving from the aqeuous phase into the hydrophobic membrane phase. This structural mechanism may be a shared motif of protein molecular switches of ...
Plants adapt to fluctuating light conditions by a process called non-photochemical quenching (NPQ... more Plants adapt to fluctuating light conditions by a process called non-photochemical quenching (NPQ), where membrane protein PsbS plays a crucial role and transforms a change in the pH-gradient across the thylakoid membrane under excess light conditions into a photoprotective state, leading to de-excitation of antenna chlorophylls. The PsbS activation mechanism is elusive and has been proposed to involve a monomerization step and protonation of specific residues. To elucidate its function, it is essential to produce PsbS in large quantities, stabilize PsbS in a membrane-mimicking environment and analyze its pH-dependent conformational structure. We present an approach for large-scale in-vitro production and spectroscopic characterization of PsbS under controlled, non-crystalline conditions. We produced PsbS of the moss Physcomitrella patens in milligram quantities in E. coli, refolded PsbS in several detergent types and analyzed its conformation at neutral and low pH by Dynamic Light ...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2017
In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII)... more In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII) collect excitations and funnel these to the photosynthetic reaction center where charge separation takes place. In excess light conditions, remodeling of the photosynthetic membrane and protein conformational changes produces a photoprotective state in which excitations are rapidly quenched to avoid photodamage. The quenched states are associated with protein aggregation, however the LHCII complexes are also proposed to have an intrinsic capacity to shift between light harvesting and fluorescence-quenched conformational states. To disentangle the effects of protein-protein and protein-lipid interactions on the LHCII photoprotective switch, we compared the structural and fluorescent properties of LHCII lipid nanodiscs and proteoliposomes with very low protein-to-lipid ratios. We demonstrate that LHCII proteins adapta fully fluorescent state in nanodiscs and in proteoliposomes with highly diluted protein densities. Increasing the protein density induces a transition into a mildly-quenched state that reaches a plateau at a molar protein-to-lipid ratio of 0.001 and has a fluorescence yield reminiscent of the light-harvesting state in vivo. The low onset for quenching strongly suggests that LHCII-LHCII attractive interactions occur inside membranes. The transition at low protein densities does not involve strong changes in the excitonic circular-dichroism spectrum and is distinct from a transition occurring at very high protein densities that comprises strong fluorescence quenching and circular-dichroism spectral changes involving chlorophyll 611 and 612, correlating with proposed quencher sites of the photoprotective mechanisms.
Photosynthetic thylakoid membranes contain the protein machinery to convert sunlight in chemical ... more Photosynthetic thylakoid membranes contain the protein machinery to convert sunlight in chemical energy and regulate this process in changing environmental conditions via interplay between lipid, protein and xanthophyll molecular constituents. This work addresses the molecular effects of zeaxanthin accumulation in thylakoids, which occurs in native systems under high light conditions through the conversion of the xanthophyll violaxanthin into zeaxanthin via the so called xanthophyll cycle. We applied biosynthetic isotope labeling and (13)C solid-state NMR spectroscopy to simultaneously probe the conformational dynamics of protein, lipid and xanthophyll constituents of thylakoids isolated from wild type (cw15) and npq2 mutant of the green alga Chlamydomonas reinhardtii, that accumulates zeaxanthin constitutively. Results show differential dynamics of wild type and npq2 thylakoids. Ordered-phase lipids have reduced mobility and mobile-phase lipids have enlarged dynamics in npq2 membra...
Journal of the American Chemical Society, Jan 8, 2015
UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used ... more UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homo-dimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time resolved fluorescence and absorption spectroscopy from femto to microseconds we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.
Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photoch... more Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photochemical quenching for photoprotection of plants under highlight conditions. The activation mechanism of PsbS and its pH-induced conformational changes are currently unknown. For structural investigation of PsbS, effective synthesis of PsbS with selective isotope or electron-spin labels or non-natural amino acids incorporated would be a great asset. This communication presents cell-free expression as a successful method for in vitro production of PsbS that would allow such incorporation. We have optimized the cell-free method to yield soluble PsbS of ~500 ng/µl using a continuous-exchange method at 30 0 C, along with a successful purification and refolding of PsbS in n-Dodecyl β-D-maltoside (β-DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.
behavior can be different within two or three cell size distance because of the chemical gradient... more behavior can be different within two or three cell size distance because of the chemical gradient in that distance. Therefore, in the modeling of such systems, a spatial resolution is required to be comparable to the cell size. Such spatial resolution poses an extra challenge in the development and execution of the computational model due to the agents sitting over multiple control volumes. In this presentation, a new computational method for cell surface-based reaction for the finite volume method will be introduced and compared to other methods in terms of accuracy and computation speed.
We investigated the temperature-mediated reassociation of the B820 subunit of Rs. rubrum to form ... more We investigated the temperature-mediated reassociation of the B820 subunit of Rs. rubrum to form a light-harvesting 1 complex (LH 1). By combining several spectroscopic techniques with global spectral data analysis fitting, we present evidence for the occurence of two spectral intermediates that appear during the reassociation process. At high temperatures, halfway the reassociation reaction, a prominent intermediate appears that has an absorption maximum around 850 nm, a fluorescence maximum around 860-867 nm, a high anisotropy (0.3 to 0.4) and a circular dichroism spectrum with three or four bands with alternating signs. At lower temperatures, more towards the end of the reassociation process, a second intermediate tends to appear that has an absorption maximum around 860 nm, a fluorescence maximum around 885 nm, a medium to high anisotropy (0.1 to 0.3) and a circular dichroism spectrum with two bands with alternating signs. The latter circular dichroism spectrum has a blueshifted...
Background: The peripheral light-harvesting complexes of photosystem II (LHCII) switch between li... more Background: The peripheral light-harvesting complexes of photosystem II (LHCII) switch between light energy capturing and dissipative states. Results: An NMR analysis is presented of the arginines in LHCII prepared in its two states. Conclusion: The protein core of arginine-glutamate interlocked helices is preserved, whereas moderate changes occur in the stromal loop region. Significance: This work reveals consequences for the molecular switching of LHCII. Light-harvesting antennae of the LHC family form transmembrane three-helix bundles of which two helices are interlocked by conserved arginine-glutamate (Arg-Glu) ion pairs that form ligation sites for chlorophylls. The antenna proteins of photosystem II have an intriguing dual function. In excess light, they can switch their conformation from a light-harvesting into a photoprotective state, in which the excess and harmful excitation energies are safely dissipated as heat. Here we applied magic angle spinning NMR and selective Arg isotope enrichment as a noninvasive method to analyze the Arg structures of the major light-harvesting complex II (LHCII). The conformations of the Arg residues that interlock helix A and B appear to be preserved in the light-harvesting and photoprotective state. Several Arg residues have very downfield-shifted proton NMR responses, indicating that they stabilize the complex by strong hydrogen bonds. For the Arg C ␣ chemical shifts, differences are observed between LHCII in the active, light-harvesting and in the photoprotective, quenched state. These differences are attributed to a conformational change of the Arg residue in the stromal loop region. We conclude that the interlocked helices of LHCII form a rigid core. Consequently, the LHCII conformational switch does not involve changes in A/B helix tilting but likely involves rearrangements of the loops and helical segments close to the stromal and lumenal ends. The elementary step in photosynthesis is the capture of solar energy by the light-harvesting antenna. In eukaryotic organisms, this step is performed by a family of pigment-binding proteins called light-harvesting complexes (LHCs) 2 that absorb sunlight and transfer the excitation energy toward the reaction centers, where charge separation takes place (1). The Lhcb antenna proteins of higher plants and moss and Lhcbm antenna proteins of LHCs in green algae contain three membrane-spanning helices A, B, and C, and their tertiary structure is typified by two crossing A/B helices interconnected by Arg-Glu ion pairs that form ligation sites for Chls. The most abundant LHC in plants and green algae is the major light-harvesting complex II (LHCII), which captures about 50% of all land-bound chlorophylls. The LHCII complex is trimeric, and each monomer forms a scaffold for eight Chla and six Chlb molecules, two luteins (Lut), one neoxanthin, and one violaxanthin, which is reversibly replaced by zeaxanthin upon de-epoxidation during the xanthophyll cycle (2-4). In addition to their light-harvesting function, LHCII complexes are involved in several regulatory mechanisms that balance the incoming excitation energies and prevent photodamage (1, 4-6). Under high sunlight conditions, the photosynthetic antenna can rapidly switch from light-harvesting into a photoprotective state in which excess light energy is safely dissipated as heat (7). This photoprotective mechanism is called non-photochemical quenching (NPQ) and protects oxygenic organisms against photooxidative damage. The major component of NPQ, qE, depends on the transmembrane proton gradient ⌬pH. In plants, a decrease in lumenal pH triggers protonation of PsbS (8, 9) and of LHCs in photosystem II (10) and activates the conversion of the LHC-bound xanthophyll cycle carotenoid from violaxanthin to zeaxanthin (11). In green algae, PsbS is absent, and the NPQ state is triggered by LhcSR, a pigment-binding complex with a short fluorescence lifetime that senses the decrease in lumenal pH (12-14). In algae, LHCII also participates in quenching, in particular the Lhcbm1 com
We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum ... more We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum rubrum from the separated R BChl 2 subunits (B820) and the oligomerization of the B820 subunit from its monomeric peptides. The full LH1 complex was reversibly associated from B820 subunits by either varying the temperature in the range 277-300 K or by varying the detergent concentration in the buffer from 0.36 to 0.52% n-octyl-D-glucopyranoside. Temperature-induced transition measurements showed hysteresis: raising the temperature induced dissociation of B873 directly into B820 subunits whereas upon recooling an intermediate spectral form was observed with an absorption maximum located around 850 nm. This intermediate form was also observed in detergent-induced transitions. It is speculated that the B850 form is a small aggregate of B820, for instance a dimer. Additionally, during a temperaturemediated transition at low detergent concentration, a set of spectral forms with maxima slightly blueshifted from 873 nm were observed, possibly due to opened rings with one or only a few R BChl 2 units missing. The temperature-induced transition of LH1 is discussed in terms of a simple assembly model. It is concluded that a moderately cooperative assembly explains the formation of small aggregates of B820 as well as of incomplete rings. Furthermore, the B820 subunits were reversibly dissociated into the monomeric B777 form by increasing either the temperature or the detergent concentration. Estimations of the enthalpy and entropy changes for the dimeric association reaction of B777 into B820 yielded an enthalpy change of-216 kJ mol-1 and an entropy change of-0.59 kJ mol-1 K-1 , at a detergent concentration of 0.8% n-octyl-D-glucopyranoside.
For the first time, data are presented on the time-resolved disassembly reaction of a highly orga... more For the first time, data are presented on the time-resolved disassembly reaction of a highly organized membrane protein complex in vitro. The photosynthetic core light-harvesting complex of the bacterial strain Rhodospirillum rubrum G9 consists of 12-16 dimeric subunits that in vivo are associated with the photosynthetic reaction center in a ringlike manner. Isolated in a detergent solution, its appearance either as a ringlike complex (called B873 and absorbing at 873 nm), subunit dimer (called B820 and absorbing at 820 nm), or monomeric form (called B777 and absorbing at 777 nm) is strongly temperaturedependent. In thermodynamic equilibria between B820 and B873, intermediate-sized complexes have also been observed that have absorption maxima around 850 nm. It is unknown whether these structures appear as intermediates in the kinetic B820-B873 (dis)assembly reaction. In this paper disassembly of the light-harvesting complex into its dimeric subunits was followed spectroscopically on a time scale up to 200 ms, upon applying an infrared laser-induced temperature jump. The full dissociation process appears to take place on a time scale of tens to hundreds of milliseconds, the rates becoming faster at higher starting temperatures. Applying the same technique, the dissociation reaction of dimeric subunits into monomers also could be established. This dissociation process occurred on a much faster time scale and took place within the 500 µs response time of our detection system.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011
The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and... more The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in 13 C-13 C dipolar homonuclear correlation spectra on a uniformly 13 C-labeled sample. In particular, we were able to resolve several chlorophyll 13 1 carbon resonances that are sensitive to hydrogen bonding to the 13 1-keto carbonyl group. The data show that 13 C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in lightharvesting regulation through MAS solid-state NMR.
In this work polarized picosecond fluorescence measurements were performed on isolated tryptophan... more In this work polarized picosecond fluorescence measurements were performed on isolated tryptophan and tryptophan in a small 22-mer peptide using a streak camera coupled to a spectrograph as a detection system. In both cases the fluorescence decay was multiexponential with decay times of ∼500 ps and ∼4 ns. Surprisingly, also a short-lived (∼16 ps) isotropic fluorescence component was found for both tryptophan and the peptide which, to our knowledge, has never been observed before. Fluorescence anisotropy data showed rotational correlation times of 155 ps and 1.5 ns for the peptide, reflecting local and overall peptide dynamics, respectively. Recently it was argued [Lakowicz, J. R. Photochem. Photobiol. 2000, 72, 421] that the nonexponential fluorescence decay of tryptophan in proteins is mainly due to spectral relaxation, whereas for isolated tryptophan it is due to different rotamers. Our results do not support this view. In contrast we conclude in both cases that the different fluorescence decay times should be ascribed to different rotameric states.
Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowe... more Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting fa...
In plant chloroplast membranes, non-photochemical quenching (NPQ) is activated as a response to a... more In plant chloroplast membranes, non-photochemical quenching (NPQ) is activated as a response to a low luminal pH and controlled by the pH-sensing protein PsbS. It has been proposed that PsbS directly interacts with the light-harvesting complexes (LHCII) of Photosystem II, inducing quenching of LHCII Chl excitations, whilst others proposed that PsbS has an indirect role in controlling the organization of the membrane. In this study, we systematically test the influence of low pH and PsbS on the fluorescence lifetimes of membrane-embedded spinach LHCII. The proteoliposome preparations contain LHCII in mild quenched states, aimed to mimic fluorescence conditions of dark-adapted leaves. We find that under those conditions, acidification and the presence of PsbS do not have significant effect on the LHCII Chl fluorescence lifetimes. This supports a view in which the functional role of PsbS consists of re-organizing the thylakoid membrane under light stress, rather than creating direct qu...
Photosynthetic light-harvesting antenna complexes (LHCs) of plants, moss and green algae form dyn... more Photosynthetic light-harvesting antenna complexes (LHCs) of plants, moss and green algae form dynamic switches between light harvesting and excitation-quenched, dissipative states. This mechanism protects the photosynthetic apparatus under light stress via a photo protective membrane response. Herein, we demonstrate the application of solid-state NMR spectroscopy to wild type, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and purified Cr Light harvesting Complex II (LHCII) reconstituted in thylakoid lipid membranes, to detect the conformational dynamics of LHCII under native conditions. We find that membrane-reconstituted LHCII contains sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Furthermore, plasticity is observed in the N-terminal stretch and in the trans-membrane helical edges facing the thylakoid lumen. In intact thylakoids, the dynamics of these protein and pigment sites is significantly reduced. We con...
The membrane protein Photosystem II subunit S (PsbS) is a pH sensor that plays an essential role ... more The membrane protein Photosystem II subunit S (PsbS) is a pH sensor that plays an essential role in signaling light stress in plants to prevent photo oxidation and generation of detrimental reactive species. PsbS detects thylakoid lumen acidification in excess light conditions via two glutamates facing the lumen, however, its molecular mechanism for activation is elusive. We performed a spectroscopic analysis of wild type Physcomitrella patens PsbS and of mutants in which the active glutamates have been replaced (E69Q, E174Q and E69Q / E174Q). We discovered that E69 exerts allosteric control of PsbS dimerization, while E174 is essential for the secondary structural response to low pH. Based on our results, we propose a molecular pH response mechanism that involves a change in the amphiphatic short helix containing E174 facing the lumen, moving from the aqeuous phase into the hydrophobic membrane phase. This structural mechanism may be a shared motif of protein molecular switches of ...
Plants adapt to fluctuating light conditions by a process called non-photochemical quenching (NPQ... more Plants adapt to fluctuating light conditions by a process called non-photochemical quenching (NPQ), where membrane protein PsbS plays a crucial role and transforms a change in the pH-gradient across the thylakoid membrane under excess light conditions into a photoprotective state, leading to de-excitation of antenna chlorophylls. The PsbS activation mechanism is elusive and has been proposed to involve a monomerization step and protonation of specific residues. To elucidate its function, it is essential to produce PsbS in large quantities, stabilize PsbS in a membrane-mimicking environment and analyze its pH-dependent conformational structure. We present an approach for large-scale in-vitro production and spectroscopic characterization of PsbS under controlled, non-crystalline conditions. We produced PsbS of the moss Physcomitrella patens in milligram quantities in E. coli, refolded PsbS in several detergent types and analyzed its conformation at neutral and low pH by Dynamic Light ...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2017
In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII)... more In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII) collect excitations and funnel these to the photosynthetic reaction center where charge separation takes place. In excess light conditions, remodeling of the photosynthetic membrane and protein conformational changes produces a photoprotective state in which excitations are rapidly quenched to avoid photodamage. The quenched states are associated with protein aggregation, however the LHCII complexes are also proposed to have an intrinsic capacity to shift between light harvesting and fluorescence-quenched conformational states. To disentangle the effects of protein-protein and protein-lipid interactions on the LHCII photoprotective switch, we compared the structural and fluorescent properties of LHCII lipid nanodiscs and proteoliposomes with very low protein-to-lipid ratios. We demonstrate that LHCII proteins adapta fully fluorescent state in nanodiscs and in proteoliposomes with highly diluted protein densities. Increasing the protein density induces a transition into a mildly-quenched state that reaches a plateau at a molar protein-to-lipid ratio of 0.001 and has a fluorescence yield reminiscent of the light-harvesting state in vivo. The low onset for quenching strongly suggests that LHCII-LHCII attractive interactions occur inside membranes. The transition at low protein densities does not involve strong changes in the excitonic circular-dichroism spectrum and is distinct from a transition occurring at very high protein densities that comprises strong fluorescence quenching and circular-dichroism spectral changes involving chlorophyll 611 and 612, correlating with proposed quencher sites of the photoprotective mechanisms.
Photosynthetic thylakoid membranes contain the protein machinery to convert sunlight in chemical ... more Photosynthetic thylakoid membranes contain the protein machinery to convert sunlight in chemical energy and regulate this process in changing environmental conditions via interplay between lipid, protein and xanthophyll molecular constituents. This work addresses the molecular effects of zeaxanthin accumulation in thylakoids, which occurs in native systems under high light conditions through the conversion of the xanthophyll violaxanthin into zeaxanthin via the so called xanthophyll cycle. We applied biosynthetic isotope labeling and (13)C solid-state NMR spectroscopy to simultaneously probe the conformational dynamics of protein, lipid and xanthophyll constituents of thylakoids isolated from wild type (cw15) and npq2 mutant of the green alga Chlamydomonas reinhardtii, that accumulates zeaxanthin constitutively. Results show differential dynamics of wild type and npq2 thylakoids. Ordered-phase lipids have reduced mobility and mobile-phase lipids have enlarged dynamics in npq2 membra...
Journal of the American Chemical Society, Jan 8, 2015
UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used ... more UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homo-dimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time resolved fluorescence and absorption spectroscopy from femto to microseconds we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.
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Papers by Anjali Pandit