Photosynthetic Membrane Proteins Research Articles

Lel A. Drachev and the direct electrometric method

In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell’ s chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.

Lel A. Drachev and the Direct Electrometric Method.

In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell's chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.

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.

Phylotranscriptomic Analyses of Mycoheterotrophic Monocots Show a Continuum of Convergent Evolutionary Changes in Expressed Nuclear Genes From Three Independent Nonphotosynthetic Lineages.

Mycoheterotrophy is an alternative nutritional strategy whereby plants obtain sugars and other nutrients from soil fungi. Mycoheterotrophy and associated loss of photosynthesis have evolved repeatedly in plants, particularly in monocots. Although reductive evolution of plastomes in mycoheterotrophs is well documented, the dynamics of nuclear genome evolution remains largely unknown. Transcriptome datasets were generated from four mycoheterotrophs in three families (Orchidaceae, Burmanniaceae, Triuridaceae) and related green plants and used for phylogenomic analyses to resolve relationships among the mycoheterotrophs, their relatives, and representatives across the monocots. Phylogenetic trees based on 602 genes were mostly congruent with plastome phylogenies, except for an Asparagales + Liliales clade inferred in the nuclear trees. Reduction and loss of chlorophyll synthesis and photosynthetic gene expression and relaxation of purifying selection on retained genes were progressive, with greater loss in older nonphotosynthetic lineages. One hundred seventy-four of 1375 plant benchmark universally conserved orthologous genes were undetected in any mycoheterotroph transcriptome or the genome of the mycoheterotrophic orchid Gastrodia but were expressed in green relatives, providing evidence for massively convergent gene loss in nonphotosynthetic lineages. We designate this set of deleted or undetected genes Missing in Mycoheterotrophs (MIM). MIM genes encode not only mainly photosynthetic or plastid membrane proteins but also a diverse set of plastid processes, genes of unknown function, mitochondrial, and cellular processes. Transcription of a photosystem II gene (psb29) in all lineages implies a nonphotosynthetic function for this and other genes retained in mycoheterotrophs. Nonphotosynthetic plants enable novel insights into gene function as well as gene expression shifts, gene loss, and convergence in nuclear genomes.

Transcriptome-wide study in the green microalga Messastrum gracile SE-MC4 identifies prominent roles of photosynthetic integral membrane protein genes during exponential growth stage

The non-model microalga Messastrum gracile SE-MC4 is a potential species for biodiesel production. However, low biomass productivity hinders it from passing the life cycle assessment for biodiesel production. Therefore, the current study was aimed at uncovering the differences in the transcriptome profiles of the microalgae at early exponential and early stationary growth phases and dissecting the roles of specific differential expressed genes (DEGs) involved in cell division during M. gracile cultivation. The transcriptome analysis revealed that the photosynthetic integral membrane protein genes such as photosynthetic antenna protein were severely down-regulated during the stationary growth phase. In addition, the signaling pathways involving transcription, glyoxylate metabolism and carbon metabolism were also down-regulated during stationary growth phase. Current findings suggested that the coordination between photosynthetic integral membrane protein genes, signaling through transcription and carbon metabolism classified as prominent strategies during exponential growth stage. These findings can be applied in genetic improvement of M. gracile for biodiesel application.

Lattice Models for Protein Organization throughout Thylakoid Membrane Stacks

Proteins in photosynthetic membranes can organize into patterned arrays that span the membrane’s lateral size. Attractions between proteins in different layers of a membrane stack can play a key role in this ordering, as was suggested by microscopy and fluorescence spectroscopy and demonstrated by computer simulations of a coarse-grained model. The architecture of thylakoid membranes, however, also provides opportunities for interlayer interactions that instead disfavor the high protein densities of ordered arrangements. Here, we explore the interplay between these opposing driving forces and, in particular, the phase transitions that emerge in the periodic geometry of stacked thylakoid membrane disks. We propose a lattice model that roughly accounts for proteins’ attraction within a layer and across the stromal gap, steric repulsion across the lumenal gap, and regulation of protein density by exchange with the stroma lamellae. Mean-field analysis and computer simulation reveal rich phase behavior for this simple model, featuring a broken-symmetry striped phase that is disrupted at both high and low extremes of chemical potential. The resulting sensitivity of microscopic protein arrangement to the thylakoid’s mesoscale vertical structure raises intriguing possibilities for regulation of photosynthetic function.

Colocalization of Autofluorescent and YFP Tagged Photosynthetic Membrane Proteins Imaged by CLSM

Colocalization of Autofluorescent and YFP Tagged Photosynthetic Membrane Proteins Imaged by CLSM

Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes.

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes invivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C.reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

Tuning the photocurrent generations from photosystem I assembled in tailored biotic-abiotic interfaces

Abstract

Interfacing Photosynthetic Membrane Protein with Mesoporous WO3 Photoelectrode for Solar Water Oxidation.

Photosynthetic biocatalysts are emerging as a new class of materials, with their sophisticated and intricate structure, which promise improved remarkable quantum efficiency compared to conventional inorganic materials in artificial photosynthesis. To break the limitation of efficiency, the construction of bioconjugated photo-electrochemical conversion devices has garnered substantial interest and stood at the frontier of the multidisciplinary research between biology and chemistry. Herein, a biohybrid photoanode of a photosynthetic membrane protein (Photosystem II (PS II)), extracted from fresh spinach entrapped on mesoporous WO3 film, is fabricated on fluorine-doped tin oxide. The PS II membrane proteins are observed to communicate with the WO3 electrode in the absence of any soluble redox mediators and sacrificial reagents under the visible light of the solar spectrum, even to 700 nm. The biohybrid electrode undergoes electron transfer and generates a significantly enhanced photocurrent compared to previously reported PS II-based photoanodes with carbon nanostructures or other semiconductor substrates for solar water oxidation. The maximum incident photon-to-current conversion efficiency reaches 15.24% at 400 nm in the visible light region. This work provides some insights and possibilities into the efficient assembly of a future solar energy conversion system based on visible-light-responsive semiconductors and photosynthetic proteins.

Exogenous spermidine maintains the chloroplast structure of cucumber seedlings and inhibits the degradation of photosynthetic protein complexes under high-temperature stress

Cucumbers (Cucumis sativus L.) are thermophilic horticulture crop but do not tolerate high temperature. The photosynthesis of cucumber under high temperature is highly susceptible to damage. To study the physiological mechanism of exogenous spermidine (Spd) on cucumber photosynthesis under heat stress, the sensitive cucumber cultivar ‘Jinchun no. 2’ was grown in substrate culture at a high temperature in an artificial climate box and treated with 1.0 mmol L−1 Spd under high-temperature stress (42/32 °C). The results showed that exogenous Spd alleviated the photosynthetic damage caused by heat shock, added the chlorophyll content and maintained the chloroplast structures relatively intact. Western blotting analysis showed that exogenous Spd inhibited the degradation of photosynthetic proteins, slowed the dissociation of the protein complexes, and maintained the stability of light-harvesting complexes (LHCs) in cucumber leaves under high-temperature stress. Twenty-two differentially expressed thylakoid membrane proteins involved in different photosynthetic processes were successfully authenticated by sodium dodecyl sulphate (SDS)-urea-PAGE and Blue native-polyacrylamide gel electrophoresis (BN-PAGE). The results showed that exogenous Spd regulated the expression of photosynthetic and bursal somatic membrane proteins, which resulted in adaptive changes under high-temperature stress at the transcriptional and translational levels, inhibited the degradation of thylakoid membrane proteins in cucumber leaves, and maintained the stable structure of the thylakoid membrane. Collectively, these results suggested that exogenous Spd alleviated high temperature-induced photosynthesis damage by improving the expression and synthesis of thylakoid membrane proteins, mitigating the dissociation of LHCII–Chl and thylakoid membrane protein complexes and maintaining the integrity and functional stability of the photosynthetic organ structure.

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.

A nanostructured cell-free photosynthetic biocomposite via molecularly controlled layer-by-layer assembly

Thylakoids, on which complete sets of photosynthetic membrane proteins are contained, behave as soft nanoparticles of a few tens nm sizes. Thus, we separated the thylakoid extracts from spinach chloroplasts and densely organized them in a film with either insulating polyethyleneimine (PEI) or conducting polyaniline (PANI) by molecularly controlled layer-by-layer (LBL) assembly. The resulting nanostructured composite films demonstrated photoelectrochemical activities with thylakoids whose stabilities were significantly improved on the charge balanced LBL multilayers compared to one on a randomly deposited film or in solution. The thylakoid LBL films continuously generated photochemical electrons for longer than 130h with the 2,6-dichlorophenolindophenol (DCPIP) mediated photosynthetic energy conversion cycle while photoactivities of thylakoid moiety slowly decreased in the open circuit potential measurements throughout which the photosynthetic redox cycles were incomplete. As the photofunctional groups of the thylakoid films originated from a biotic system and the photofuctional thylakoids were exceptionally well preserved on the charge-balanced artificial structures, the developed thylakoid LBL film have shown inherent biocompatibility upon PC12 neural cell attachment and differentiation tests, which are essential qualities for emerging human-friendly electronic applications such as disposable electronics, artificial retina, and implantable neural interfacing meditronics as well as cell-free photosynthetic production.

New ultrarapid-scanning interferometer for FT-IR spectroscopy with microsecond time-resolution.

A novel Fourier-transform infrared (FT-IR) rapid-scan spectrometer has been developed (patent pending EP14194520.4) which yields 1000 times higher time resolution as compared to conventional rapid-scanning spectrometers. The central element to achieve faster scanning rates is based on a sonotrode whose front face represents the movable mirror of the interferometer. A prototype spectrometer with a time resolution of 13 μs was realized, capable of fully automated long-term measurements with a flow cell for liquid samples, here a photosynthetic membrane protein in solution. The performance of this novel spectrometer is demonstrated by recording the photoreaction of bacteriorhodopsin initiated by a short laser pulse that is synchronized to the data recording. The resulting data are critically compared to those obtained by step-scan spectroscopy and demonstrate the relevance of performing experiments on proteins in solution. The spectrometer allows for future investigations of fast, non-repetitive processes, whose investigation is challenging to step-scan FT-IR spectroscopy.

Biomimetic wiring and stabilization of photosynthetic membrane proteins with block copolymer interfaces

Block copolymer membranes stabilize photosystem I in a long-lasting, high performance photocurrent generating device.

Identification of genes involved in color variation of bamboo culms by suppression subtractive hybridization

Phyllostachys vivax cv. aureocaulis is a widely planted ornamental bamboo with evergreen leaves. This plant's culm exhibits a golden-yellow background color marked randomly with narrow and broad green stripes but is occasionally light green with yellow stripes. In this study, we attempt to identify the molecular mechanism underlying the color variation in striped culms. We found that neither stroma nor grana lamellas were observed in plastids in yellow tissue cells, while complete chloroplasts were observed in green tissue. In addition, chlorophyll a and b were mainly distributed in ground tissue under the epiderm and in the cells surrounding the bundle sheath in the green portion of internodes. The amount of chlorophyll contained in cross-sections of the green portion of culms is significantly higher than in the yellow portion. However, carotenoid was nearly undetectable in both samples. In addition, we found that the expression levels of 7 ESTs, including PvESTs-F641 (JZ893845), PvESTs-F681 (JZ893885) and PvESTs-F798 (JZ894002), were significantly higher in green samples than that in yellow samples, while PvESTs-R200 (JZ894906), PvESTs-R541 (JZ895247), PvESTs-R333 (JZ895039) and PvESTs-R266 (JZ894972) were found at a higher level in yellow samples. These ESTs are thought to play a role in this color variation in plants. Our current results indicate that insufficient photosynthetic membrane proteins and lipids in yellow tissue could lead to chloroplast dysfunction and could result in the yellow appearance on certain P. vivax cv. aureocaulis culms.

Interference lithographic nanopatterning of plant and bacterial light-harvesting complexes on gold substrates.

We describe a facile approach for nanopatterning of photosynthetic light-harvesting complexes over macroscopic areas, and use optical spectroscopy to demonstrate retention of native properties by both site-specifically and non-specifically attached photosynthetic membrane proteins. A Lloyd's mirror dual-beam interferometer was used to expose self-assembled monolayers of amine-terminated alkylthiolates on gold to laser irradiation. Following exposure, photo-oxidized adsorbates were replaced by oligo(ethylene glycol) terminated thiols, and the remaining intact amine-functionalized regions were used for attachment of the major light-harvesting chlorophyll–protein complex from plants, LHCII. These amine patterns could be derivatized with nitrilotriacetic acid (NTA), so that polyhistidine-tagged bacteriochlorophyll–protein complexes from phototrophic bacteria could be attached with a defined surface orientation. By varying parameters such as the angle between the interfering beams and the laser irradiation dose, it was possible to vary the period and widths of NTA and amine-functionalized lines on the surfaces; periods varied from 1200 to 240 nm and linewidths as small as 60 nm (λ/4) were achieved. This level of control over the surface chemistry was reflected in the surface topology of the protein nanostructures imaged by atomic force microscopy; fluorescence imaging and spectral measurements demonstrated that the surface-attached proteins had retained their native functionality.

Simultaneous refolding of denatured PsbS and reconstitution with LHCII into liposomes of thylakoid lipids.

The thylakoid membrane protein PsbS is critical for quenching excessive excitation energy in mechanisms that involve the light-harvesting complexes of photosystem II. Liposomes of thylakoid lipids have been shown to be a very good platform to study photosynthetic membrane proteins and their interactions. In this study, we simultaneously refolded and reconstituted functional pea PsbS into liposomes of thylakoid lipids starting from denatured expressed protein. Intrinsic fluorescence spectroscopy, trypsin digestion, and circular dichroism spectroscopy were used to characterize the native state of PsbS in the proteoliposomes. The functionality of refolded PsbS was further demonstrated by its effect on the fluorescence quenching of the major antenna system of photosystem II (LHCII) co-inserted into the liposomes. The fluorescence yield of native trimeric LHCII was lowered by PsbS by 50% at neutral pH and by a further 25% upon lowering the pH to 4.5. Furthermore, the acid-induced fluorescence reduction was completely reversed by addition of N,N'-dicyclohexylcarbodiimide, an inhibitor of protein protonation. These results indicate that reconstituted PsbS induces strong quenching of LHCII sensing changes in local pH via its protonation sites.

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.