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The energy transfer dynamics of the biohybrid photosynthetic antenna complexes were investigated by transient absorption. The mechanism of energy transfer was discussed in terms of the higher-lying exciton state of bacteriochlorophyll aggregates.

The two-dimensional molecular assembly was accomplished of bacteriochlorophyll

Photosynthetic light-harvesting (LH) systems consist of photosynthetic pigments, which are non-covalently self-assembled with protein scaffolds in many phototrophs and attain highly efficient excitation energy transfer via ultrafast dynamics. In this study, we constructed a biohybrid LH system composed of an LH complex (LH2) from Rhodoblastus acidophilus strain 10050 and a hydrophobic fluorophore ATTO647N (ATTO) as an extrinsic antenna in the lipid bilayer. Through the addition of ATTOs into a solution of LH2-reconstituted lipid vesicles, ATTOs were incorporated into the hydrophobic interior of the lipid bilayer to configure the non-covalently self-assembled biohybrid LH. Steady-state fluorescence spectroscopy clearly showed efficient energy transfer from ATTO to B850 bacteriochlorophylls in LH2. Femtosecond transient absorption spectroscopy revealed that the energy transfer took place in the time range of 3–13 ps, comparable to that of the covalently linked LH2-ATTO that we previously reported. In addition, the biohybrid LH system exhibited a much higher antenna effect than the LH2-ATTO system because of the higher loading level of ATTO in the membrane. These findings suggest that the facile self-assembled biohybrid LH system is a promising system for constructing LH for solar-energy conversion.

A functional linkage between light-harvesting and photocatalytic components is a pivotal issue for using solar energy in chemical conversions; however, this concept is far from being practically re...

Phycobilisomes (PBSs) are photosynthetic antenna megacomplexes comprised of pigment-binding proteins (cores and rods) joined with linker proteins. A rod-type PBS that does not have a core is connected to photosystem I (PSI) by a pigment-free CpcL linker protein, which induces a red-shift of the absorption band of phycocyanobilin (PCB) in the rod (red-PCB). Herein, the isolated supercomplex of the rod-type PBS and the PSI tetramer from Anabaena sp. PCC 7120 were probed by picosecond laser spectroscopy at 77 K and by decay-associated spectral analysis to show that red-PCB mediates the fast (time constant = 90 ps) and efficient (efficiency = 95%) transfer of excitation energy from PCB in rod to chlorophyll a (Chl a) in PSI. According to the Förster energy transfer mechanism, this high efficiency corresponds to a 4-nm distance between red-PCB and Chl a, suggesting that β-84 PCB in rod acts as red-PCB.TOC GRAPHIC

The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (kon, koff) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that kon and koff of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. koff decreased by increasing the amount of amine groups in the NP, whereas kon did not change. Importantly, NPs showing a low koff at pH 5.5 together with a high koff at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.

A small interfering RNA (siRNA) delivery system using dioleylphosphate-diethylenetriamine conjugate (DOP-DETA)-based liposomes (DL) was assessed for systemic delivery of siRNA to tumors. DL carrying siRNA capable of inducing efficient gene silencing with low doses of siRNA were modified with polyethylene glycol (PEG-DL/siRNA) for systemic injection of siRNA. The biodistribution of DL and siRNA in the PEG-DL/siRNA was studied by using radiolabeled DL and fluorescence-labeled siRNA, respectively. DL in the PEG-DL/siRNA showed a high retention in the plasma, accumulation in the tumor, and low accumulation in the liver and spleen after intravenous injection. The in vivo effects of PEGylation were observed only when distearoylphosphatidylethanolamine (DSPE)-PEG but not distearoylglycerol (DSG)-PEG were used. This result suggests that the electrostatic interaction between lipid molecules on the surface of PEG-DL/siRNA was a critical determinant for the in vivo effect of PEGylation. When PEG-DL/siRNA (0.1 mg/kg siRNA) was intravenously injected into tumor-bearing mice, in vivo gene silencing was observed in subcutaneous tumors. These results indicate that PEG-DL/siRNA designed in this study is a promising formulation for systemic use of siRNA.

While the influence of pKa provided by amine-containing materials in siRNA delivery vectors for use in gene-silencing has been widely studied, there are little reports in which amine pKa is controlled rigorously by using bioisosteres and its effect on gene-silencing. Here, we report that amine pKa could be rigorously controlled by replacement of hydrogen atom(s) with fluorine atom(s). A series of mono- and di-amine lipids with a different number of fluorine atoms were synthesized. The pKa of the polyamine lipids was shifted to a lower value with an increase in the number of fluorine atoms. The optimal pKa for high gene-silencing efficiency varied according to the number of amine residues in the polyamine lipid. Whereas the endosomal escape ability of mono-amine lipid-containing lipid vesicles (LVs) depended on the pKa, that of all tested di-amine lipid-containing LVs showed equal membrane-destabilizing activity. LVs showing moderately weak interactions with siRNA facilitated cytoplasmic release of siRNA, resulting in strong gene-silencing. These findings indicate that appropriate amine pKa engineering depending on the number of amines is important for the induction of effective RNA interference.

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C

Photosynthesis in natural systems is one of the typical examples where the interaction between multiple photons and multiple chromophores leads to the synergetic responses in three-dimensionally arranged molecular aggregates. Accordingly, the investigation of primary events in the excited state could provide important information on the progress in the artificial photosynergetic systems. Along with this line, we have prepared biohybrid light-harvesting complexes to study ultrafast dynamics of excitation energy transfer in the present project. Light-harvesting 2 complex (LH2) from photosynthetic purple bacteria is a molecular assembly, in which multiple photosynthetic chromophores are well organized. To expand high-harvesting activity of LH2, we attached a fluorophore, Alexa647, to lysine residues on the LH2 polypeptide so as to cover broad solar spectrum. Ultrafast energy transfer from Alexa 647 to the intrinsic chromophores B800 and B850 in LH2 was observed using femtosecond transient absorption spectroscopy. Detailed analysis revealed two possible energy transfer pathways. In addition, we made a LH2 mutant bearing reactive Cys residue on the N-terminal region of LH2β polypeptide. The engineered LH2 allows us to provide the defined position of Alexa647. Together with the system in which LH2 was dissolved in micellar solution, a lipid bilayer system in which the bioengineered LH2 was incorporated was applied to control the location of the external fluorophore.

The heterostructures fabricated with a combination of 2-D materials and conventional semiconductors are of great interest owing to their unique optoelectronic properties. Here, we demonstrate the fabrication of a germanium (Ge)/MoS2 heterostructure and observation of a photovoltaic action with broadband photoresponsivity. A thin interface was formed in-between the thermally evaporated Ge and chemical vapor deposited MoS2 layers, considering the atomically smooth surface of MoS2. The current density-voltage ( ${J}$ – ${V}$ ) measurement showed a diode like characteristic and photoresponsivity under light illumination. The Ge/MoS2 heterojunction showed an open circuit voltage ( ${V}_{\text {oc}}$ ) of 0.185 V and short circuit current density ( ${J}_{\text {sc}}$ ) of 0.028 mA/cm2 under illumination of light (wavelength 350–1100 nm). The transient photoresponse characteristics of the Ge-MoS2 heterojunction device showed significant photoresponsivity for ultraviolet, visible, and near-infrared light at different bias voltages. The interfacial charge transfer at the Ge-MoS2 heterojunction is the critical aspect to create an effective build in field for the broadband photoresponsive device. The demonstrated ultrathin heterojunction of Ge and MoS2 layers can be significant for developing self-powered broadband photodetectors.

With the aim of understanding the excitation energy transfer mechanism in natural photosynthetic membranes, light-harvesting (LH)2 and LH1-reaction center, which are pigment-protein complexes separated from Rhodobacter sphaeroides, were aligned on a planar electrode surface in stripe patterns at 5 urn intervals. Observation of the absorption spectrum and fluorescence microphotographs revealed selective immobilization and conservation of the pigments. Photocurrent signals were obtained when the electrode was illuminated at either 880 or 800 nm. The fabricated structure was confirmed to function as a natural photosynthetic membrane with the highest photocurrent signal being obtained when using a co-immobilized substrate under excitation at 800 nm.

The photosynthetic light-harvesting–reaction center core complex (LH1-RC) is a natural excitonic and photovoltaic device embedded in a lipid membrane. In order to apply LH1-RCs as a biohybrid energy-producing material, some important issues must be addressed, including how to make LH1-RCs function as efficiently as possible. In addition, they should be characterized to evaluate how many active LH1-RCs efficiently work in artificial systems. We report here that an anionic phospholipid, phosphatidylglycerol (PG), stabilizes the charge-separated state (a photooxidized electron donor and reduced quinone pair, P+QB–) of LH1-RC (from Rhodopseudomonas palustris) and enhances its activity in photocurrent generation. Steady-state fluorometric analysis demonstrated that PG enhances the formation of the P+QB– state at lower irradiances. The photocurrent generation activity was analyzed via Michaelis–Menten kinetics, revealing that 38% of LH1-RCs reconstituted into the PG membrane generated photocurrent at a turnover...

Enhancing the light-harvesting potential of antenna components in a system of solar energy conversion is an important topic in the field of artificial photosynthesis. We constructed a biohybrid light-harvesting complex 2 (LH2) engineered from Rhodobacter sphaeroides IL106 strain. An artificial fluorophore Alexa Fluor 647 maleimide (A647) was attached to the LH2 bearing cysteine residue at the N-terminal region (LH2-NC) near B800 bacteriochlorophyll a (BChl) assembly. The A647-attached LH2-NC conjugate (LH2-NC-A647) preserved the integrity of the intrinsic chromophores, B800- and B850-BChls, and carotenoids. Femtosecond transient absorption spectroscopy revealed that the sequential energy transfer A647 → B800 → B850 occurs at time scale of 9–10 ps with monoexponential dynamics in micellar and lipid bilayer systems. A B800-removed conjugate (LH2-NC[B800(−)]-A647) exhibited a significant decrease in energy transfer efficiency in the micellar system; however, surprisingly, direct energy transfer from A647 to B850 was observed at a rate comparable to that for LH2-NC-A647. This result implies that the energy transfer pathway is modified after B800 removal. The results obtained suggested that a LH2 complex is a potential platform for construction of biohybrid light-harvesting materials with simple energy transfer dynamics through the site-selective attachment of the external antennae and the modifiable energy-funnelling pathway.

Lipid-based nanoparticles, a potential nonviral vector due to their good biocompatibility and biodegradability, have been extensively developed for the delivery of small interfering RNA (siRNA). We designed a unique pH-responsive lipid derivative, a dioleylphosphate-diethylenetriamine conjugate (DOP-DETA). DOP-DETA consists of a pH-responsive triamine and unsaturated fatty acids that accelerate membrane fusion. Our results showed that DOP-DETA-based liposomes (DL) efficiently delivered siRNA into the cytoplasm and induced RNA interference even at a low siRNA concentration. The knockdown efficiency of DL depended on the molar ratio of total DL lipids to siRNA. When siRNA was formulated with a sufficient amount of DL, it was efficiently taken up by cells and induced effective gene silencing. Time-lapse imaging showed that siRNA transfected with DL was rapidly internalized into the cells and uniformly dispersed in the cytoplasm within a few minites. The results also showed that DL induced sufficient change in surface charge to allow it to interact with the cell membrane and to allow for rapid endosomal escape. Uptake pathway and time-lapse imaging studies revealed that siRNA was delivered by DL into the cytoplasm, possibly through both macropinocytosis and membrane fusion. The present results emphasize that the modulation of surface charge on nanoparticles is crucial for each siRNA delivery process. Our results also suggest that DL is a potentially useful vector for inducing gene silencing with low-doses of siRNA.

Light-harvesting complex of photosystem II (LHCII) is the most abundant membrane protein-chlorophyll complex in chloroplasts. Here, we evaluated the photocatalytic activity of the native trimer and the enzymatically treated monomer forms of LHCII. Upon white light irradiation using a solar simulator, photocatalytic reduction of methylviologen revealed that the activity of monomeric LHCII was higher than that of the trimer form. Fluorescence of the monomeric LHCII was more significantly quenched by methylviologen than that of the trimer form. In the presence of Pt nanoparticles in a catalytic solution system, photoinduced hydrogen production was observed for the LHCII monomer. The difference in photocatalytic activities of LHCII trimer and monomer are briefly discussed in terms of an interaction between LHCII and methylviologen.

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C18-monoacetylenic (9-octadecynyl) chains, DF8CCH8PC, were compared with those of its non-fluorinated counterpart, DH8CCH8PC. Light-harvesting complex 2 (LH2) and the light-harvesting 1‒reaction center core complex (LH1-RC) isolated from purple photosynthetic bacteria were employed as probe membrane proteins to evaluate the extent to which their reconstitution into DF8CCH8PC membranes could proceed. DF8CCH8PC formed more expanded and more stable fluid monolayers than DH8CCH8PC at the air-water interface at 25 °C; the former PC molecule occupied an area of ca. 0.70 nm2 at a collapse pressure, πc, of 52 mN/m, while the latter occupied an area of ca. 0.55 nm2 at a πc of 45 mN/m. In contrast, the molecular motion detected using fluorescent probes was much more restricted in DF8CCH8PC bilayers than in DH8CCH8PC ones. Although the reconstitution efficiencies of both LH2 and LH1-RC into DF8CCH8PC bilayers were lower than those into DH8CCH8PC bilayers, the membrane proteins incorporated into DF8CCH8PC bilayers showed increased thermostability. The increased thermostability of these proteins in fluorinated PC membranes might be due to the restricted molecular motion in the hydrophobic chains. The results of this study suggest that partially fluorinated PCs can be useful materials for the construction of lipid‒functional membrane protein assemblies including large membrane protein complexes, such as LH1-RC, for biotechnological applications.

A light harvesting system that effectively drives photocatalytic component is essential for converting solar light into chemical energy. Here we addressed the extension of light harvesting activity and presented a quantitative analysis of the functional linkage between light harvesting and a photocatalytic reaction of biohybrid light-harvesting 1–reaction center core complex (LH1-RC from Rhodopseudomonas palustris). Light harvesting wavelengths were extended by a series of fluorophores (Alexa647, 680, 750, and ATTO647N), which were covalently attached to LH1-RC (LH1-RC-Fluors). Femtosecond transient absorption spectroscopy revealed ultrafast excitation energy transfer (EET) from the fluorophores to bacteriochlorophyll a pigments in LH1 (B875), followed by charge separation in RC. The rate of EET increased with increase in the spectral overlap between the emission bands of the fluorophores and the absorption band of B875. The attachment of the external light harvesting fluorophores boosted the maximum yield of charge separation and photocurrent generation activity. We discussed the effects of the number of fluorophores attached to LH1-RC and location of the fluorophores in a lipid bilayer environment on light harvesting activity.

We successfully developed novel high molecular weight (Mw) solubilization surfactants for membrane proteins, Bis-D 3 -DKDKC 12 (Mw ∼3 kDa), Bis-K 3 -DKDKC 12 (Mw ∼3 kDa), and Tris-D 3 -DKDKC 12 (Mw ∼4.3 kDa), which were rationally designed from our previously established peptide gemini surfactants (PG-surfactants) DKDKC 12 K and DKDKC 12 D (Mw ∼1.3 kDa). An increase in Mw enhanced affinity between membrane proteins and surfactants, thereby allowing effective solubilization, even for lower concentration ranges (

To develop efficient systems for light-energy conversion, monomeric and dimeric zinc chlorin derivatives were synthesized. These derivatives act as photosensitizers for light-induced hydrogen production with methylviologen as the electron mediator and Pt nanoparticles as the hydrogen-evolution catalyst. The monomeric derivative exhibited ~1.5-times higher activity than the dimeric derivative. The photocurrent-generating activity of the Zn chlorin derivatives assembled on a chemically modified indium tin oxide electrode was investigated; the dimeric derivative exhibited significant activity compared to the monomeric derivative. The photosensitizing activity of the derivatives in dye-sensitized solar cells was also investigated. The dimeric derivative exhibited two-fold higher performance than the monomeric derivative. The dimerization effect on the photosensitizing activities is briefly discussed in terms of stacking conformations based on the results of absorption and fluorescence spectroscopies.

In recent years, a number of light-induced hydrogen production systems composed of photosystem I (PSI) and hydrogen production catalysts (e.g. hydrogenases and Pt nanoparticles) have been reported. However, the utility of these systems under aerobic conditions is limited due to their poor stability in the presence of oxygen. The development of light-induced hydrogen production systems that work under aerobic conditions is, therefore, of great importance to establish artificial photosynthetic devices. Ideally, these systems should utilise water as an electron source, via water splitting by photosystem II (PSII). We report the construction of a novel light-induced hydrogen production system composed of PSI-platinum nanoparticle conjugates and cytochrome c 6 (cyt c 6) immobilised in nanoporous glass plates (PGP50, 50-nm pore diameter). PSI trimer (PSIt) from Thermosynechococcus elongatus and Pt nanoparticles (PtNPs) were conjugated via electrostatic interactions (PSIt-PtNP). PSIt-PtNP and cyt c 6 were spontaneously absorbed in nanopores of PGP50 without denaturation. Upon irradiation in the presence of ascorbate as a sacrificial electron donor, catalytic H2 evolution was observed for PSIt-PtNP immobilised in the pores of PGP50 (PSIt-PtNP/PGP50) under both anaerobic and aerobic conditions, indicating that an effective photoinduced electron transfer system had been established. PSIt-PtNP/PGP50 was found to exhibit improved oxygen resistivity over the homogeneous solution system consisting of PSIt-PtNP, cyt c 6, and ascorbate, suggesting that the PSIt-PtNP/PGP50 system could be a potential candidate for artificial photosynthetic systems. The distribution of the components, PSIt-PtNP and cyt c 6, in PGP50 was characterised to discuss the efficiency of light-induced hydrogen production.

The development of artificial photosynthesis has focused on the efficient coupling of reaction at photoanode and cathode, wherein the production of hydrogen (or energy carriers) is coupled to the electrons derived from water-splitting reactions. The natural photosystem II (PSII) complex splits water efficiently using light energy. The PSII complex is a large pigment-protein complex (20 nm in diameter) containing a manganese cluster. A new photoanodic device was constructed incorporating stable PSII purified from a cyanobacterium Thermosynechococcus vulcanus through immobilization within 20 or 50 nm nanopores contained in porous glass plates (PGPs). PSII in the nanopores retained its native structure and high photoinduced water splitting activity. The photocatalytic rate (turnover frequency) of PSII in PGP was enhanced 11-fold compared to that in solution, yielding a rate of 50-300 mol e(-)/(mol PSII·s) with 2,6-dichloroindophenol (DCIP) as an electron acceptor. The PGP system realized high local concentrations of PSII and DCIP to enhance the collisional reactions in nanotubes with low disturbance of light penetration. The system allows direct visualization/determination of the reaction inside the nanotubes, which contributes to optimize the local reaction condition. The PSII/PGP device will substantively contribute to the construction of artificial photosynthesis using water as the ultimate electron source.

Morphology of dipalmitoyl phosphatidyl choline (DPPC)-cholesterol (Chol) mixed monolayer formed on water surface by dropping method was investigated using surface tension measurement (STm), Brewster angle microscopy (BAM), and fluorescence microscopy (FM). STm showed strong condensation effect of Chol in fluidic DPPC monolayer. Excess area (Sex) from mean mixing state of DPPC and Chol was about twice larger than that by general compression method in the range from xC = 0.2 to 0.4 (xC: mole fraction of Chol). BAM and FM images showed clearly that the fluidic DPPC monolayer changed to condensed rigid monolayer due to the condensation effect of Chol. At more than xC = 0.3 DPPC-Chol mixed monolayer changed to condensed state similar to the Chol monolayer. These results support previous reports by compression method that Chol molecule demonstrates the strong condensation effect to the fluidic monolayer and also indicate that dropping method enables to form unique monolayer on the water surface.

While the influence of pK

The photosynthetic light-harvesting-reaction center core complex (LH1-RC) is a natural excitonic and photovoltaic device embedded in a lipid membrane. In order to apply LH1-RCs as a biohybrid energy-producing material, some important issues must be addressed, including how to make LH1-RCs function as efficiently as possible. In addition, they should be characterized to evaluate how many active LH1-RCs efficiently work in artificial systems. We report here that an anionic phospholipid, phosphatidylglycerol (PG), stabilizes the charge-separated state (a photooxidized electron donor and reduced quinone pair, P

Introducing appropriate artificial components into natural biological systems could enrich the original functionality. To expand the available wavelength range of photosynthetic bacterial light-harvesting complex 2 (LH2 from Rhodopseudomonas acidophila 10050), artificial fluorescent dye (Alexa Fluor 647: A647) was covalently attached to N- and C-terminal Lys residues in LH2 α-polypeptides with a molar ratio of A647/LH2 ≃ 9/1. Fluorescence and transient absorption spectroscopies revealed that intracomplex energy transfer from A647 to intrinsic chromophores of LH2 (B850) occurs in a multiexponential manner, with time constants varying from 440 fs to 23 ps through direct and B800-mediated indirect pathways. Kinetic analyses suggested that B800 chromophores mediate faster energy transfer, and the mechanism was interpretable in terms of Förster theory. This study demonstrates that a simple attachment of external chromophores with a flexible linkage can enhance the light harvesting activity of LH2 without affecting inherent functions of energy transfer, and can achieve energy transfer in the subpicosecond range. Addition of external chromophores, thus, represents a useful methodology for construction of advanced hybrid light-harvesting systems that afford solar energy in the broad spectrum.

Zinc chlorophyll a derivatives were synthesised for use as photosensitizers. Herein, we report a system for photoinduced hydrogen production with colloidal platinum via photoreduction of methylviologen (MV2+) using the photosensitization of a Zn pyropheophorbide a (ZnPyOH) dimer connected by lysine (ZnPy-K(ZnPy)OH) as a special pair model in the photosynthetic reaction centre in the presence of NADPH as an electron donor.

PTEN-positive tumors are not susceptible to the treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR). Here, we determined the susceptibility of PTEN-positive cells to small interfering RNA for mTOR (si-mTOR) by using a novel liposomal delivery system. We prepared dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL) decorated with polyethylene glycol (PEG) grafting Ala-Pro-Arg-Pro-Gly (APRPG), a VRGFR-1-targeting peptide. APRPG-PEG-decorated TEPA-PCL carrying si-mTOR (APRPG-TEPA-PCL/si-mTOR) had an antiproliferative effect against B16F10 murine melanoma cells (PTEN-positive) and significantly inhibited both the proliferation and tube formation of mouse 2H-11 endothelial-like cells (PTEN-positive). APRPG-TEPA-PCL/si-mTOR treatment did not induce Akt phosphorylation (Ser473) in either B16F10 or 2H-11 cells although there was strong phosphorylation of Akt in response to rapamycin treatment. Intravenous injection of APRPG-TEPA-PCL/si-mTOR significantly suppressed the tumor growth compared with rapamycin treatment in mice bearing B16F10 melanoma. These findings suggest that APRPG-TEPA-PCL/si-mTOR is useful for the treatment of PTEN-positive tumors.

Appropriate experimental platforms are required to clarify the structure–function relationships of membrane protein assemblies. In photosynthetic bacteria, light-harvesting complex 2 and light-harvesting/reaction center core complex play key roles in capturing and transferring light energy and facilitating subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly in the photosynthetic membrane. However, the mechanism through which this assembly influences the efficiency of energy conversion remains to be clarified. We review our recent studies that were conducted to evaluate the structure–function relationship of the supramolecular assembly of photosynthetic antenna complexes in various lipid bilayer systems, as well as the construction of novel systems of planar lipid membranes for use as experimental platforms.

Photosystem II (PSII) has attracted a lot of attention for use in the construction of artificial photosynthetic materials due to its high activity of oxidation of water molecules. However, the robustness of PSII needs to be improved for in vitro application. In this study, we incorporated PSII (Thermosynechococcus vulcanus) into various phospholipid membranes to examine the activity and durability of oxygen evolution. PSII was incorporated into anionic 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (PSII-DOPG), zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (PSII-DOPC), and cationic 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (PSII-EDOPC). Structural integrity of PSII was examined by absorption and fluorescence spectroscopy. Compared with PSII dissolved in a micellar solution of n-dodecyl-β-d-maltoside (PSII-micelle), durability of PSII-DOPC and PSII-DOPG were enhanced by 1.3- and 1.5-fold, respectively. The activity and durability of PSII-EDOPC was significantly low. Lipid-dependent activity and durability were discussed in terms of kinetic parameters of V max and K m, and inhibition of the electron acceptor, phenyl-p-benzoquinone.

Photosystems, PSI and PSII isolated from Thermosynechococcus elongatus were successfully immobilized on a TiO2 nanostructured film for use in dye-sensitized biosolar cells (DSBCs). The photosystem complexes were also immobilized on an ITO electrode modified with 3-aminopropyltriethoxysilane by utilizing the interactions between the electrode and the surface of the PSI or PSII polypeptide. Illumination of the PSI and PSII complexes immobilized on the ITO electrode resulted in action spectra in the presence of methyl viologen, which corresponded to the absorption spectra of the complexes. Compared with the ITO electrode, PSI or PSII complexes assembled on the TiO2 electrode had much higher energy-conversion efficiency in the presence of an iodide/triiodide redox system of an ionic-liquid-based electrolyte. This could have interesting applications in the development of DSBCs.

Hydrogenases are powerful catalysts for light-driven H2 production using a combination of photosensitizers. However, except oxygen-tolerant hydrogenases, they are immediately deactivated under aerobic conditions. We report a light-driven H2 evolution system that works stably even under aerobic conditions. A [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F was immobilized inside nanoporous glass plates (PGPs) with a pore diameter of 50 nm together with a ruthenium complex and methyl viologen as a photosensitizer and an electron mediator, respectively. After immersion of PGP into the medium containing the catalytic components, an anaerobic environment automatically established inside the nanopores even under aerobic external conditions upon irradiation with solar-simulated light; this system constantly evolved H2 with an efficiency of 3.7 μmol H2 m(-2) s(-1). The PGP system proposed in this work represents a promising first step toward the development of an O2-tolerant solar energy conversion system.

We herein report the synthesis and characterization of unique chemically reactive solubilization surfactants, Alk-DKDKC12K and Bis-alk-DKDKC12K that consist of the DKDKC12K unit, previously reported to function as a novel class of lipopeptide-based solubilization surfactant for membrane proteins such as photosystem I (PSI) derived from cyanobacteria, and one or two alkyne groups at the N- and/or C-terminal sides, which could react with bis-azide crosslinker via Huisgen cycloaddition reaction. By a stepwise in situ crosslinking of PSI solubilized in a buffer, containing Alk-DKDKC12K and Bis-alk-DKDKC12K, with a set of crosslinkers (the first one is bis azide compounds and the second is formylated dextran), we succeeded in preparing hydrogel microfibers encapsulating PSI without damage to the original photophysical functions.

Background We previously developed a microRNA (miRNA) delivery system by using dicetyl phosphate-tetraethylenepentamine-based polycation liposomes (TEPA-PCL), applied it to miR-92a delivery, and demonstrated its gene-silencing potential and effective anti-angiogenic effects. In the present study, we investigated the mechanism of intracellular delivery of cholesterol-grafted miR-92a (miR-92a-C) into cells. Methods To investigate the intracellular distribution of miR-92a-C/TEPA-PCL complex, we used human umbilical vein endothelial cells and examined certain points after transfection: (i) the time-course of miR-92a-uptake into the cells; (ii) the endocytosis pathway induced by miR-92a-C/TEPA-PCL; (iii) the capability of miR-92a-C/TEPA-PCL to escape from the endosomes; and (iv) the release of miR-92a-C from TEPA-PCL in the cytoplasm. Results Our data indicated that miR-92a-C formulated in TEPA-PCL accumulated in and was spread throughout the cytoplasm in a time-dependent manner, and was taken up into the cells by macropinosome-mediated endocytosis. In addition, the surface charge of miR-92a-C/TEPA-PCL was neutral at pH 7.4 and was charged positively at around pH 5.5, which is the inner pH of endosomes. When the late endosomes/lysosomes were stained with Lysotracker, miR-92a-C/TEPA-PCL efficiently escaped from the endosomes into the cytoplasm, possibly through the proton-sponge effect. Furthermore, miR-92a-C spread throughout whole cytoplasm and did not co-localize completely with TEPA-PCL, indicating that some of the miR-92a-C was present in free form in the cytoplasm. Conclusions The results of the present study suggest that TEPA-PCL-based lipoplexes have an excellent potential to deliver microRNAs into the cytoplasm of cells and to induce RNA silencing action mediated by microRNAs and other small RNAs. Copyright © 2013 John Wiley & Sons, Ltd.

In the photosynthetic membrane of purple bacteria networks of light-harvesting 2 (LH2) complexes capture the sunlight and transfer the excitation energy. In order to investigate the mutual relationship between the supramolecular organization of the pigment-protein complexes and their biological function, the LH2 complexes were reconstituted into three types of phospholipid membranes, consisting of L-α-phosphatidylglycerol (PG), L-α-phosphatidylcholine (PC), and L-α-phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Atomic force microscopy (AFM) revealed that the type of phospholipids had a crucial influence on the clustering tendency of the LH2 complexes increased from PG over PC to PE/PG/CL, where the LH2 complexes formed large, densely packed clusters. Time-resolved spectroscopy uncovered a strong quenching of the LH2 fluorescence that is ascribed to singlet-singlet and singlet-triplet annihilation by an efficient energy transfer between the LH2 complexes in the artificial membrane systems. Quantitative analysis reveals that the intercomplex energy transfer efficiency varies strongly as a function of the morphology of the nanostructure, namely in the order PE/PG/CLPCPG, which is in line with the clustering tendency of LH2 observed by AFM. These results suggest a strong influence of the phospholipids on the self-assembly of LH2 complexes into networks and concomitantly on the intercomplex energy transfer efficiency.

In purple photosynthetic bacteria, light-harvesting complex 2 (LH2) and light harvesting/reaction centre core complex (LH1-RC) play the key roles of capturing and transferring light energy and subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly; however, how the assembly influences the efficiency of energy conversion is not yet clear. We addressed this issue by evaluating the energy transfer in reconstituted photosynthetic protein complexes LH2 and LH1-RC and studying the structures and the membrane environment of the LH2/LH1-RC assemblies, which had been embedded into various lipid bilayers. Thus, LH2 and LH1-RC from Rhodopseudomonas palustris 2.1.6 were reconstituted in phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE)/PG/cardiolipin (CL). Efficient energy transfer from LH2 to LH1-RC was observed in the PC and PE/PG/CL membranes. Atomic force microscopy revealed that LH2 and LH1-RC were heterogeneously distributed to form clusters in the PC and PE/PG/CL membranes. The results indicated that the phospholipid species influenced the cluster formation of LH2 and LH1-RC as well as the energy transfer efficiency.

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials.

Preparation of DPPC lipid monolayer in water trough has been done by dropping method and compared with compression method. Monolayer was studied by surface pressure isotherm, fluorescence microscopy, Brewster angle microscopy, and infrared external reflection spectroscopy. Results of these measurements showed that dropping method gave better results compared to compression method. In dropping method, transition from liquid expanded state to liquid condensed is gradual compared to sharp one in compressed method. During monolayer formation, adjustment and interaction between hydrophilic part of lipid and water and among hydrophobic part of lipid molecule are slow, stable, and more natural as worked out from surface area versus pressure isotherm. At a given molecular area, surface pressure is less compared to compression method thus monolayer is in more fluidic state in dropping method than compression method. The observation was supported by all techniques described above.

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.

The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membran...

Genetic therapy using microRNA-499 (miR-499) was combined with chemotherapy for the advanced treatment of cancer. Our previous study showed that miR-499 suppressed tumor growth through the inhibition of vascular endothelial growth factor (VEGF) production and subsequent angiogenesis. In the present study, we focused on blood flow in tumors treated with miR499, since some angiogenic vessels are known to lack blood flow. Tetraethylenepentamine-based polycation liposomes (TEPA-PCL) were prepared and modified with Ala-Pro-Arg-Pro-Gly peptide (APRPG) for targeted delivery of miR-499 (APRPG-miR-499) to angiogenic vessels and tumor cells. The tumor blood flow was significantly improved, so-called normalized, after systemic administration of APRPG-miR-499 to Colon 26 NL-17 carcinoma–bearing mice. In addition, the accumulation of doxorubicin (DOX) in the tumors was increased by pre-treatment with APRPG-miR-499. Moreover, the combination therapy of APRPG-miR-499 and DOX resulted in significant suppression of the tumors. Taken together, our present data indicate that miR-499 delivered with APRPG-modified-TEPA-PCL normalized tumor vessels, resulting in enhancement of intratumoral accumulation of DOX. Our findings suggest that APRPG-miR-499 may be a therapeutic, or a combination therapeutic, candidate for cancer treatment.