Photosynthetic Membrane Protein Complexes Research Articles

Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells.

Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.

生体エネルギー変換にかかわる生体超分子複合体の構造研究

Three-dimensional protein structures bring us a deeper insight into the biological function. The main aim of my group is the X-ray structure determination of the biological macromolecular assemblies including membrane protein complexes in order to elucidate the molecular mechanism of the highly organized biological processes at atomic level. Here, I report two examples; One is structural studies of photosynthetic membrane protein complex and related redox enzymes, and the other is crystal structure analyses of dynein motor.

The Use of Advanced Mass Spectrometry to Dissect the Life-Cycle of Photosystem II.

Photosystem II (PSII) is a photosynthetic membrane-protein complex that undergoes an intricate, tightly regulated cycle of assembly, damage, and repair. The available crystal structures of cyanobacterial PSII are an essential foundation for understanding PSII function, but nonetheless provide a snapshot only of the active complex. To study aspects of the entire PSII life-cycle, mass spectrometry (MS) has emerged as a powerful tool that can be used in conjunction with biochemical techniques. In this article, we present the MS-based approaches that are used to study PSII composition, dynamics, and structure, and review the information about the PSII life-cycle that has been gained by these methods. This information includes the composition of PSII subcomplexes, discovery of accessory PSII proteins, identification of post-translational modifications and quantification of their changes under various conditions, determination of the binding site of proteins not observed in PSII crystal structures, conformational changes that underlie PSII functions, and identification of water and oxygen channels within PSII. We conclude with an outlook for the opportunity of future MS contributions to PSII research.

Photosynthetic membrane organization and role of state transition in cyt, cpII, stt7 and npq mutants of Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, cytochrome b6/f and chlorophyll b binding proteins are important in energy distribution between photosystem (PS)II and PSI. In this study, we have used C. reinhardtii mutants deficient in cytochrome b6/f complex (cyt), chlorophyll b binding protein (cpII), non-photochemical quenching (npq) and LHC II kinase (stt7) to study the importance of these proteins in electron transport, phosphorylation, and structural organization of thylakoid supercomplexes under optimum growth conditions. Fast Chl a fluorescence studies have shown that lack of CpII and Cyt b6/f caused reduced photochemical yield (Fv/Fm). The disappearance of I phase in cyt mutant showed that electron transfer from Cyt b6/f to PSI is reduced due to un availability of Q0 site for docking of PQH2 therefore LHC II kinase was unable to phosphorylate LHCII in cyt mutant. Further, blue native gel electrophoresis revealed the differential organization of photosynthetic membrane protein complexes in different mutants. Particularly, LHCII trimerization is more in cyt and stt7 mutants. Also, chemically induced state transition (LHCII phosphorylation) was not observed in cyt mutant, however, all other mutants were similar to that of wild type. Based on our results, we propose that the LHCII trimer accumulation and its organization with other complexes are very important in state transitions.

Triton X-100 as an effective surfactant for the isolation and purification of photosystem I from Arthrospira platensis

Surfactants play important roles in the preparation, structural, and functional research of membrane proteins, and solubilizing and isolating membrane protein, while keeping their structural integrity and activity intact is complicated. The commercial n-Dodecyl-β-D-maltoside (DDM) and Triton X-100 (TX) were used as solubilizers to extract and purify trimeric photosystem I (PSI) complex, an important photosynthetic membrane protein complex attracting broad interests. With an optimized procedure, TX can be used as an effective surfactant to isolate and purify PSI, as a replace of the much more expensive DDM. A mechanism was proposed to interpret the solubilization process at surfactant concentrations lower than the critical solubilization concentration. PSI-TX and PSI-DDM had identical polypeptide bands, pigment compositions, oxygen consumption, and photocurrent activities. This provides an alternative procedure and paves a way for economical and large-scale trimeric PSI preparation.

A Novel Photosynthetic Strategy for Adaptation to Low-Iron Aquatic Environments

Iron (Fe) availability is a major limiting factor for primary production in aquatic environments. Cyanobacteria respond to Fe deficiency by derepressing the isiAB operon, which encodes the antenna protein IsiA and flavodoxin. At nanomolar Fe concentrations, a PSI-IsiA supercomplex forms, comprising a PSI trimer encircled by two complete IsiA rings. This PSI-IsiA supercomplex is the largest photosynthetic membrane protein complex yet isolated. This study presents a detailed characterization of this complex using transmission electron microscopy and ultrafast fluorescence spectroscopy. Excitation trapping and electron transfer are highly efficient, allowing cyanobacteria to avoid oxidative stress. This mechanism may be a major factor used by cyanobacteria to successfully adapt to modern low-Fe environments.

The Assembly of a Multisubunit Photosynthetic Membrane Protein Complex: A Site-Specific Spin Labeling EPR Spectroscopic Study of the PsaC Subunit in Photosystem I

The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site-specific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored from the perspective of a reporter spin label attached to either the native C34(C) or the engineered C75(C) residue of wild-type PsaC (PsaC(WT)). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P(700)-F(X)/PsaC complex, and the P(700)-F(X)/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaC(WT) binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P(700)-F(X)/PsaC(WT-C34) complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I core. The binding of several PsaC variants, each lacking one or more key binding contacts with the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34(C). Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaC(C-term)) causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P(700)-F(X)/PsaC(C-term-C34) complex. Because the PsaC(C-term) variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180 degrees-rotated, C(2)-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core.

Peptidyl–Prolyl Isomerase Activity in Chloroplast Thylakoid Lumen is a Dispensable Function of Immunophilins in Arabidopsis thaliana

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2% of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.

Magic angle spinning (MAS) NMR: a new tool to study the spatial and electronic structure of photosynthetic complexes

In the last two decades, Magic Angle Spinning (MAS) NMR has created its own niche in studies involving photosynthetic membrane protein complexes, owing to its ability to provide structural and functional information at atomic resolution of membrane proteins when in the membrane, in the natural environment. The light-harvesting two (LH2) transmembrane complex from Rhodopseudomonas acidophila is used to illustrate the procedure of the technique applicable in photosynthesis research. One- and two-dimensional solid-state NMR experiments involving 13C- and 15N-labeled LH2 complexes allow to make a sequence-specific assignment of NMR signals, which forms the basis for resolving structural details and the assessment of charge transfer, electronic delocalization effects, and functional strain in the ground state.

Conformational Flexibility in Assembly of Photosynthetic Membrane Protein Complexes in vivo

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

VOCs – helping trees withstand global change?

VOCs – helping trees withstand global change?

Rebinding of the extrinsic proteins of Photosystem II studied by electron microscopy and single particle alignment: an assessment with small two-dimensional ordered arrays of Photosystem II

Structural analysis of weakly ordered two-dimensional (2-D) ordered arrays of Photosystem II (a photosynthetic membrane protein complex) has been carried out using single-particle alignment and averaging (SPA). Although this averaging technique is normally applied to randomly oriented solubilised particles, we have found that it is also highly effective for the analysis of membrane-located 2-D ordered arrays and that the arrays can be used to check the effectiveness of the rotational alignment routines. Successful rotational alignment varied in the range 75 to 66% and 26 to 19%, depending on the stain used and hence the signal-to-noise ratio, and the data were used to address questions of space group assignments for Photosystem II 2-D ordered arrays. We thus hoped to settle the controversy as to the oligomeric form of PS II in native thylakoids, i.e., the monomeric vs. dimeric model. The methods were applied to the study of the removal and rebinding of extrinsic subunits in Photosystem II. Information on the location of the subunits in this large membrane protein complex is presented here.

Novel methodology for topological studies of photosynthetic membrane protein complexes

AbstractNovel techniques have‐been developed for the investigation of photosynthetic membrane protein complexes from the thylakoid membrane of spinach. The protein components of photosystem II were fractionated by a solid phase extraction procedure using Chromabond cartridges. The cytochrome b6f complex was dissociated with high concentrations of the nonionic detergent n‐dodecyl‐β‐D‐maltoside. Subsequently the subunits of both membrane protein complexes were separated by high resolution reversed phase HPLC. Nearest neighbour relationships were investigated in the cytochrome b6f complex by molecular cross‐linking with o‐phthaldialdehyde. A methodology has been developed to identify the cross‐linking sites on the molecular level by tryptic digestion of the protein conjugates and separation of the fragments by high resolution HPLC. Matrix Assisted Laser Desorption Ionization‐Mass Spectrometry (MALDI‐MS) was used as a sensitive tool to identify the protein components and crosslinked peptide fragments of photosystem II and the cytochrome b6f complex.

Molecular sizes of photosynthetic membrane-protein complexes

Molecular sizes of photosynthetic membrane-protein complexes