Chlorophyll-binding Proteins Research Articles

C-Terminal M4 Peptide Fragment of Albino 3 Interaction with cpSRP-43

The protein complexes responsible for insertion of light harvesting chlorophyll binding protein into the thylakoid membrane of the chloroplast are the concern of this investigation. Critical for this insertion, the membrane bound protein Albino 3 has been shown to bind a cpSRP-FTSY-LHCP complex. The following study implicates the stromal, c-terminus of Albino 3 as an imperative binding interaction in this process. Isothermal titration calorimetry and 1H-15N HSQC were used to understand the binding of a 16 residue segment from this region of Albino 3 known as M4. The data show that this segment binds with 14uM affinity and that electrostatic interactions with E327 and E356 on the 43kDa subunit of cpSRP are essential for this event. Importantly, significant binding is not observed when two domains of cpSRP-43, CD2 and CD3, are separated. Far UV and intrinsic fluorescence experiments also show that, upon M4 binding, moderate conformational changes in CD2-CD3 occur.

Investigation of the Structural Stability of cpSRP43 Chromodomain2 by Hydrogen-Deuterium Exchange

The essential pathway by which light-harvesting chlorophyll binding proteins are inserted into the thylakoid of the chloroplast is mediated by a unique signal recognition particle (cpSRP). Its novel 43kDa subunit (cpSRP43) contains three chromodomains (CDs). CD2 has been shown to be significant in various aspects of the cpSRP-mediated pathway. While structural information about CD2 is known, this study aims to gain a more comprehensive understanding of the structural stability through analysis of hydrogen-deuterium exchange. 15N-labeled CD2 samples will be produced from over-expression of CD2 in E. coli cultured in labeled media. Subsequently, the readily-occurring exchange between amide hydrogen and solvent deuterium will be monitored by NMR spectroscopy. The kinetics of the exchange can provide useful information about the free energy of exchange and thus the stability.

Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish

Hydrophilic chlorophyll (Chl)-binding proteins have been isolated from various Brassicaceae plants and are categorized into Class II water-soluble Chl-binding proteins (WSCPs). Although the molecular properties of class II WSCPs including Brassica-type (e.g., cauliflower WSCP, Brussels sprouts WSCP and BnD22, a drought- and salinity-stress-induced 22kDa protein of rapeseed), a Lepidium-type, and an Arabidopsis-type WSCPs have been well characterized, those of Raphanus-type WSCPs are poorly understood. To gain insight into the molecular diversity of Class II WSCPs, we cloned a novel cDNA encoding a Raphanus sativus var. raphanistroides (Japanese wild radish called ‘Hamadaikon’) WSCP (RshWSCP). Sequence analysis revealed that the open reading frame of the RshWSCP gene consisted of 666bp encoding 222 aa residues, including 23 residues of a deduced signal peptide. Functional recombinant RshWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (RshWSCP-His). Although the RshWSCP-His was expressed as a soluble protein in E. coli, the apo-protein was highly unstable and tended to aggregate during a series of purification steps. When the soluble fraction of RshWSCP-His-expressing E. coli was mixed immediately with homogenate of spinach leaves containing thylakoid, RshWSCP-His was able to remove Chl molecules from the thylakoid and formed a stable Chl-WSCP complex with high hydrophilicity. UV–visible absorption spectra of the reconstituted RshWSCP-His revealed that RshWSCP-His is one of the Class IIA WSCP with the highest Chl a/b ratio analyzed thus far. A semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that RshWSCP was transcribed in buds and flowers but not in roots, stems and various leaves.

The search for new chlorophyll-binding proteins in the cyanobacterium Synechocystis sp. PCC 6803

Light harvesting provides a major challenge in the production of biofuels from microorganisms; while sunlight provides the energy necessary for biomass/biofuel production, at the same time it damages the cells. The genome of Synechocystis sp. PCC 6803 was searched for open reading frames that might code for yet unidentified chlorophyll-binding proteins with low molecular mass that could be involved in stress-adaptation. Amongst 9167 hypothetical ORFs corresponding to potential polypeptides of 100 amino acids or less, two were identified that had the potential to be pigment-binding, because they (i) encoded a potential transmembrane region, (ii) showed sequence similarity with known chlorophyll-binding domains, (iii) were conserved in other cyanobacterial species, and (iv) their codon adaptation index indicated significant translation probability. The two ORFs were located complementary (antisense) and internal to the ferrochelatase (hemH) and the pyruvate dehydrogenase (pdh) genes and therefore were named a-fch and a-pdh, respectively. Transcription of both genes was confirmed; however, no translated proteins could be detected immunologically. Whereas mutations within a-pdh or a-fch did not lead to any obvious phenotype, it is clear that transcripts and proteins over and above the currently known set may play a role in defining the physiology of cyanobacteria and other organisms.

The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection

Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII.

Abundances of Iron-Binding Photosynthetic and Nitrogen-Fixing Proteins of Trichodesmium Both in Culture and In Situ from the North Atlantic

Marine cyanobacteria of the genus Trichodesmium occur throughout the oligotrophic tropical and subtropical oceans, where they can dominate the diazotrophic community in regions with high inputs of the trace metal iron (Fe). Iron is necessary for the functionality of enzymes involved in the processes of both photosynthesis and nitrogen fixation. We combined laboratory and field-based quantifications of the absolute concentrations of key enzymes involved in both photosynthesis and nitrogen fixation to determine how Trichodesmium allocates resources to these processes. We determined that protein level responses of Trichodesmium to iron-starvation involve down-regulation of the nitrogen fixation apparatus. In contrast, the photosynthetic apparatus is largely maintained, although re-arrangements do occur, including accumulation of the iron-stress-induced chlorophyll-binding protein IsiA. Data from natural populations of Trichodesmium spp. collected in the North Atlantic demonstrated a protein profile similar to iron-starved Trichodesmium in culture, suggestive of acclimation towards a minimal iron requirement even within an oceanic region receiving a high iron-flux. Estimates of cellular metabolic iron requirements are consistent with the availability of this trace metal playing a major role in restricting the biomass and activity of Trichodesmium throughout much of the subtropical ocean.

Line Narrowing of Excited-State Transitions in Nonlinear Polarization Spectroscopy: Application to Water-Soluble Chlorophyll-Binding Protein

The homogeneous linewidth of dye aggregates like photosynthetic light-harvesting complexes contains important information about energy transfer and relaxation times that is, however, masked by inhomogeneous broadening caused by static disorder. Whereas there exist line narrowing techniques for the study of low-energy exciton states, the homogeneous linewidth of the high-energy states is not so easy to decipher. Here we present a microscopic theory for nonlinear polarization spectroscopy in the frequency domain that contains a dynamic aggregate selection revealing the homogeneous linewidth of these states. The theory is applied to the water-soluble chlorophyll-binding protein for which the high-energy exciton state was predicted to exhibit a sub-100-fs lifetime.

AtACDO1, an ABC1-like kinase gene, is involved in chlorophyll degradation and the response to photooxidative stress in Arabidopsis

ABC1 (activity of bc1 complex) is a newly discovered atypical kinase in plants. Here, it is reported that an ABC1 protein kinase-encoded gene, AtACDO1 (ABC1-like kinase related to chlorophyll degradation and oxidative stress), located in chloroplasts, was up-regulated by methyl viologen (MV) treatment. AtACDO1 RNAi (RNA interference) plants showed developmental defects, including yellow-green leaves and reduced contents of carotenoids and chlorophyll; the chlorophyll reduction was associated with a change in the numbers of chlorophyll-binding proteins of the photosynthetic complexes. Chlorophyllide (Chlide) a the first product of chlorophyll degradation, and pheophorbide a, a subsequent intermediate of Chlide a degradation, were increased in AtACDO1 RNAi plants. The AtACDO1 RNAi plants were more sensitive to high light and MV than wild-type plants. The AtACDO1 RNAi plants had lower transcript levels of the oxidative stress response genes FSD1, CSD1, CAT1, and UTG71C1 after MV treatment compared with wild-type or 35S::AtACDO1 plants. Taken together, the results suggest that the chloroplast AtACDO1 protein plays important roles in mediating chlorophyll degradation and maintaining the number of chlorophyll-binding photosynthetic thylakoid membranes, as well as in the photooxidative stress response.

Phylogenetic analysis of the light-harvesting system in Chromera velia

Chromera velia is a newly discovered photosynthetic eukaryotic alga that has functional chloroplasts closely related to the apicoplast of apicomplexan parasites. Recently, the chloroplast in C. velia was shown to be derived from the red algal lineage. Light-harvesting protein complexes (LHC), which are a group of proteins involved in photon capture and energy transfer in photosynthesis, are important for photosynthesis efficiency, photo-adaptation/accumulation and photo-protection. Although these proteins are encoded by genes located in the nucleus, LHC peptides migrate and function in the chloroplast, hence the LHC may have a different evolutionary history compared to chloroplast evolution. Here, we compare the phylogenetic relationship of the C. velia LHCs to LHCs from other photosynthetic organisms. Twenty-three LHC homologues retrieved from C. velia EST sequences were aligned according to their conserved regions. The C.velia LHCs are positioned in four separate groups on trees constructed by neighbour-joining, maximum likelihood and Bayesian methods. A major group of seventeen LHCs from C. velia formed a separate cluster that was closest to dinoflagellate LHC, and to LHC and fucoxanthin chlorophyll-binding proteins from diatoms. One C. velia LHC sequence grouped with LI1818/LI818-like proteins, which were recently identified as environmental stress-induced protein complexes. Only three LHC homologues from C. velia grouped with the LHCs from red algae.

The SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LCHB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis in Arabidopsis seedlings

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons. SCO2 encodes a chloroplast-localized protein disulphide isomerase, hypothesized to be involved in protein folding. Analysis of co-expressed genes with SCO2 revealed that genes with similar expression patterns encode chloroplast proteins involved in protein translation and in chlorophyll biosynthesis. Indeed, sco2-1 accumulates increased levels of the chlorophyll precursor, protochlorophyllide, in both dark grown cotyledons and leaves. Yeast two-hybrid analyses demonstrated that SCO2 directly interacts with the chlorophyll-binding LHCB1 proteins, being confirmed in planta using bimolecular fluorescence complementation (BIFC). Furthermore, ultrastructural analysis of sco2-1 chloroplasts revealed that formation and movement of transport vesicles from the inner envelope to the thylakoids is perturbed. SCO2 does not interact with the signal recognition particle proteins SRP54 and FtsY, which were shown to be involved in targeting of LHCB1 to the thylakoids. We hypothesize that SCO2 provides an alternative targeting pathway for light-harvesting chlorophyll binding (LHCB) proteins to the thylakoids via transport vesicles predominantly in cotyledons, with the signal recognition particle (SRP) pathway predominant in rosette leaves. Therefore, we propose that SCO2 is involved in the integration of LHCB1 proteins into the thylakoids that feeds back on the regulation of the tetrapyrrole biosynthetic pathway and nuclear gene expression.

Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates

Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn(4)Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K(63)↔CP43D(321) (trypsin) and CP43K(215)↔Psb27D(58)AGGLK(63)↔CP43D(321) (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.

Plants switch photosystem at high temperature to protect photosystem II

AbstractPlants are often exposed to temperatures of around 40^o^C. These temperatures can cause serious damage to photosystems, yet plants can survive with minimum damage. Here, we show that plants switch photosystem to protect photosystem II (PSII) at 40^o^C. Using wheat and Arabidopsis seedlings, we investigated the mechanisms of heat-derived damage in the dark and avoidance of damage in the light. Heat treatment at 40^o^C in the dark caused serious damage to PSII: the maximum quantum yield of PSII (Fv/Fm) and oxygen evolution rapidly decreased. The damage was due to the degradation of the D1 protein (shown by immuno-chemical analysis) and the disturbance of energy transfer in PSII core chlorophyll-binding proteins CP43 and CP47 (shown by time-resolved fluorescence measurement). The damage to PSII might be due to enhanced introduction of electrons from the reducing power of the stroma into thylakoid membranes, causing subsequent electron backflow to PSII. Plants treated at 40^o^C in the light avoided PSII damage and showed preferential excitation of photosystem I (PSI), phosphorylation and migration of light-harvesting complex II (LHCII), which indicate state transition of the photosystem to enhance thermal dispersion and light-driven cyclic electron flow around PSI. These results suggest that heat damage to PSII is probably due to a backflow of reducing power from the stroma to PSII, and that light causes a state transition of photosystem, driving cyclic electron flow and thus protecting PSII from damage.

Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43–substrate interaction domain

A cpSRP [chloroplast SRP (signal recognition particle)] comprising cpSRP54 and cpSRP43 subunits mediates the insertion of light-harvesting proteins into the thylakoid membrane. We dissected its interaction with a full-length membrane protein substrate in aqueous solution by insertion of site-specific photo-activatable cross-linkers into in vitro-synthesized Lhcb1 (major light-harvesting chlorophyll-binding protein of photosystem II). We show that Lhcb1 residues 166-176 cross-link specifically to the cpSRP43 subunit. Some cross-link positions within Lhcb1 are in the 'L18' peptide required for targeting of cpSRP substrates, whereas other cross-linking positions define a new targeting signal in the third transmembrane span. Lhcb1 was not found to cross-link to cpSRP54 at any position, and cross-linking to cpSRP43 is unaffected by the absence of cpSRP54. cpSRP43 thus effectively binds substrates autonomously, and its ability to independently bind an extended 20+-residue substrate region highlights a major difference with other SRP typeswhere the SRP54 subunit binds to hydrophobic target sequences. The results also show that cpSRP43 can bind to a hydrophobic, three-membrane span, substrate in aqueous solution, presumably reflecting a role for cpSRP in the chloroplast stroma. This mode of action, and the specificity of the cpSRP43-substrate interaction, may be associated with cpSRP's unique post-translational mode of action.

One-step isolation and biochemical characterization of a highly active plant PSII monomeric core.

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009).

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway

Higher plants require chloroplasts for essential functions in photosynthesis and other important physiological processes, such as sugar, lipid and amino-acid biosynthesis. Most chloroplast proteins are nuclear-encoded proteins that are synthesized in the cytosol as precursors, and imported into chloroplasts by protein translocases in the outer and inner chloroplast envelope. The imported chloroplast proteins are then translocated into or across the thylakoid membrane by four distinct pathways. However, the mechanisms by which the imported nuclear-encoded proteins are delivered to these pathways remain largely unknown. Here we show that an Arabidopsis ankyrin protein, LTD (mutation of which causes the light-harvesting chlorophyll-binding protein translocation defect), is localized in the chloroplast and using yeast two-hybrid screens demonstrate that LTD interacts with both proteins from the signal recognition particle (SRP) pathway and the inner chloroplast envelope. Our study shows that LTD is essential for the import of light-harvesting chlorophyll-binding proteins and subsequent routing of these proteins to the chloroplast SRP-dependent pathway.

Functions of the water soluble chlorophyll-binding protein in plants

Functional aspects of water soluble chlorophyll-binding protein (WSCP) in plants were investigated during the courses of leaf senescence, chlorophyll biogenesis, stress response and photoprotection. The cDNA sequence encoding WSCP from cauliflower was cloned into a binary vector to facilitate Agrobacterium tumefaciens mediated transformation of Nicotiana tabacum. The resultant transgenic tobacco plants overexpressed the CauWSCP gene under the control of a 35S-promoter. Analyses of protein and pigment contents indicate that WSCP overexpression does not enhance chlorophyll catabolism in vivo, thus rendering a role of WSCP in Chl degradation unlikely. Accumulation of higher levels of protochlorophyllide in WSCP overexpressor plants corroborates a proposed temporary storage and carrier function of WSCP for chlorophyll and late precursors. Although WSCP overexpressor plants did not show significant differences in non-photochemical quenching of chlorophyll fluorescence, they are characterized by significantly lower zeaxanthin accumulation and peroxidase activity at different light intensities, even at high light intensities of 700–900 μmol photons m −2 s −1. These results suggest a photoprotective function of the functional chlorophyll binding-WSCP tetramer by shielding of chlorophylls from molecular oxygen.

Excitonic Energy Level Structure and Pigment−Protein Interactions in the Recombinant Water-Soluble Chlorophyll Protein. I. Difference Fluorescence Line-Narrowing

Difference fluorescence line-narrowing spectroscopy at 4.5 K was employed to investigate electron-phonon and electron-vibrational coupling strengths of the lower exciton level of water-soluble chlorophyll-binding protein (WSCP) from cauliflower reconstituted with chlorophyll a or chlorophyll b, respectively. The electron-phonon coupling is found to be moderate with integral Huang-Rhys factors S in the order of 0.81-0.85. A weak dependence of S on excitation wavelength within the inhomogeneously broadened fluorescence origin band is attributed to a sizable contribution of nonresonant excitation that varies with excitation wavelength. The strongly asymmetric and highly structured one-phonon profile is characterized by a peak phonon frequency (ω(m)) of ~24 cm(-1) and further discernible peaks at 48 and 88 cm(-1), respectively. A structural assignment of this unusual one-phonon profile is proposed. As will be shown in the accompanying paper (part II) (DOI 10.1021/jp111457t), the parameters of electron-phonon coupling readily account for shape and position of the fluorescence origin bands at 666.1 and 683.8 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. A rich structure of S(1)→S(0) vibrational frequencies was resolved in the wavenumber range between 180 and 1665 cm(-1) for both chlorophyll a- and chlorophyll b-WSCP. The corresponding individual Huang-Rhys factors fall in the range between 0.0011 and 0.0500. To the best of our knowledge, this is the first report of S-factors for vibrational modes of chlorophyll b. Most remarkable is the presence of two additional modes at 228 and 327 cm(-1) compared with the vibrational spectrum of chlorophyll in solution. The additional modes can most likely be attributed to H-bond formation in the vicinity of the chlorophyll molecule bound by WSCP.

Excitonic Energy Level Structure and Pigment−Protein Interactions in the Recombinant Water-Soluble Chlorophyll Protein. II. Spectral Hole-Burning Experiments

Persistent spectral hole burning at 4.5 K has been used to investigate the excitonic energy level structure and the excited state dynamics of the recombinant class-IIa water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The hole-burned spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) at the burn wavelength, (ii) a number of vibrational ZPHs, (iii) a broad low-energy hole at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, and (iv) a second satellite hole at ~658 and ~673 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The doublet of broad satellite holes is assigned to an excitonically coupled chlorophyll dimer. The lower-energy holes at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, represent the lower exciton states. Taking into account the parameters of electron-phonon coupling, the lower exciton state can be assigned as the fluorescence origin. The lower exciton state is populated by two processes: (i) exciton relaxation from the higher exciton state and (ii) vibrational relaxation within the lower exciton state. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm(-1) for chlorophyll b- and chlorophyll a-WSCP, respectively, based on the positions of the satellite holes. The Gaussian low-energy absorption band identified by constant fluence hole burning at 4.5 K has a width of ~150 cm(-1) and peaks at 664.9 and 682.7 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The action spectrum is broader and blue-shifted compared to the fluorescent lower exciton state. This finding can be explained by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (J. Phys. Chem. B2008, 112, 13951).

Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies

In higher plants the light energy is captured by the photosynthetic pigments that are bound to photosystem I and II and their light-harvesting complex (LHC) subunits. In this study, we examined the photodynamic changes within chlorophyll-protein complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Chlorophyll-protein complexes were isolated using sucrose density gradient centrifugation and blue-native polyacrylamid gel electrophoresis (BN-PAGE). Proteome analysis was performed using SDS-PAGE, HPLC and high resolution mass spectrometry. We identified several rarely expressed and stress-induced chlorophyll-binding proteins, showed changes in localization of early light-induced protein family and LHC protein family members between different photosynthetic complexes and assembled/disassembled subcomplexes under light stress conditions and discuss their role in a variety of light stress-related processes.

A Dynamic cpSRP43-Albino3 Interaction Mediates Translocase Regulation of Chloroplast Signal Recognition Particle (cpSRP)-targeting Components*

The chloroplast signal recognition particle (cpSRP) and its receptor, chloroplast FtsY (cpFtsY), form an essential complex with the translocase Albino3 (Alb3) during post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCPs). Here, we describe a combination of studies that explore the binding interface and functional role of a previously identified cpSRP43-Alb3 interaction. Using recombinant proteins corresponding to the C terminus of Alb3 (Alb3-Cterm) and various domains of cpSRP43, we identify the ankyrin repeat region of cpSRP43 as the domain primarily responsible for the interaction with Alb3-Cterm. Furthermore, we show Alb3-Cterm dissociates a cpSRP·LHCP targeting complex in vitro and stimulates GTP hydrolysis by cpSRP54 and cpFtsY in a strictly cpSRP43-dependent manner. These results support a model in which interactions between the ankyrin region of cpSRP43 and the C terminus of Alb3 promote distinct membrane-localized events, including LHCP release from cpSRP and release of targeting components from Alb3.