Energy Transfer on Cytoskeleton of Red Blood Cell Ghosts and their Efficiency Control by KCl Concentration-induced Cell Shrinkage.

Lipid bilayer membranes such as liposomes have been utilized as platforms for bioinspired artificial photosynthesis. Embedding functional compounds, including chromophores and catalysts, into two-dimensional lipid membranes allows their high local concentration and proximity, resulting in enhanced reactivity compared to that of homogeneous solutions. The control of photoreactions by the physical and chemical properties of membranes, such as fluidity and phase separation, has also been well studied in recent years. In contrast, it remains difficult to control chemical reactions via dynamic membrane deformation. Here, we report on the control of excitation energy transfer using red blood cell ghosts (RBCGs) as scaffolds, relying on their asymmetric lipid membranes and inherent and unique deformability. RBCGs, in which donor and acceptor molecules were chemically conjugated to a two-dimensional cytoskeleton located beneath the inner membrane, exhibited energy transfer, and their efficiency varied depending on the amount and ratio of donor and acceptor modifications, as confirmed by experimental and theoretical analysis. Furthermore, the KCl concentration-induced RBCG shrinkage enhanced the energy transfer efficiency. Our proposed method is expected to facilitate the construction of photoreaction systems that can be controlled via membrane deformation.

Cu(II)-Triggered Ion Channel Properties of a 2,2'-Bipyridine-Modified Amphotericin B.

The development of stimuli-responsive synthetic channels that open and close in response to physical and chemical changes in the surrounding environment has attracted attention because of their potential bioapplications such as sensing, drug release, antibiotics, and molecular manipulation tools to control membrane transport in cells. Metal coordination is ideal as a stimulus for stimuli-responsive channels because it allows for reversible gating behavior through the addition and removal of metal ions and fine-tuning of channel structure through coordination geometry defined by the type of the metal ion and ligand. We have previously reported on transition metal-ion dependent ion permeability control of Amphotericin B (AmB) modified with a metal coordination site, 2,2'-bipyridine ligand (bpy-AmB). AmB is one of the polyene macrolide antibiotics, and it is known that the interaction between AmB and ergosterol molecules is required for AmB channel formation. In contrast, the Cu2+ coordination to the bpy moiety of bpy-AmB induces formation of Ca2+ ion-permeable channels in the ergosterol-free POPC membrane. However, the details of bpy-AmB properties such as channel stability, ion selectivity, pore size, and the effect of ergosterol on channel formation remain unclear. Here, we investigate bpy-AmB channels triggered by transition metal coordination in POPC or ergosterol-containing POPC liposomes using an HPTS assay, electrophysiological measurements, and time-resolved UV-vis spectral measurements. These analyses reveal that bpy-AmB channels triggered by Cu2+ ions are more stable and have larger pore sizes than the original AmB channels and enable efficient permeation of various cations. We believe that our channel design will lead to the construction of metal coordination-triggered synthetic ion channels.

Peptide modification on the interior surface of red blood cell ghosts for construction of catalytic reactors.

We have succeeded in in situ synthesis of gold nanoparticles (Au NPs) on the interior surfaces of red blood cell ghosts (RBCGs) with a cytoskeleton chemically conjugated to a gold-binding peptide. The Au NP-deposited RBCG with the peptide exhibits enhanced catalytic reactivity for the reduction of 4-nitrophenol due to the uniform distribution of the Au NPs on the inner surface and the coverage of the Au NPs surface with the peptide.

pH-Dependent ion permeability control of a modified amphotericin B channel through metal complexation.

Amphotericin B incorporating 2,2'-bipyridine (bpy-AmB) forms a membrane channel exhibiting pH-dependent Ca2+ ion permeability with a selective response to Cu2+ ions. The coordination structure at bpy sites depends on the pH and metal ions can control the association state of bpy-AmB in the membrane.

Nonanuclear Ni(ii) complexes in a [1-7-1] formation derived from asymmetric multidentate ligands: magnetic and electrochemical properties.

Nonanuclear Ni(ii) complexes, [Ni9(Ln)6(OH)6(H2O)6] (Ni9Ln, n = 1-4; H2Ln = 6-acetoacetyl-2-pyridinecarboxylic acid derivatives), were prepared via self-assembly using the asymmetric multidentate ligands H2Ln. A corner-sharing tetrahedron-type structure, [Ni7(µ3-OH)6]8+, and terminal mononuclear units constitute the nonanuclear structure in a [1-7-1] formation. The electrochemical and magnetic properties of Ni9Ln were modulated by the introduction of various substituents in H2Ln.

Direct Synthesis of Prussian Blue Nanoparticles in Liposomes Incorporating Natural Ion Channels for Cs+ Adsorption and Particle Size Control.

Coordination polymer (CP) nanoparticles (NPs) formed by a self-assembly of organic ligands and metal ions are one of the attractive materials for molecular capture and deliver/release in aqueous media. Control of particle size and prevention of aggregation among CP NPs are important factors for improving their adsorption capability in water. We demonstrate here the potential of a liposome incorporating an antibiotic ion channel as a vessel for synthesizing Prussian blue (PB) NPs, being a typical CP. In the formation of PB NPs within liposomes, the influx rate of Fe2+ ions into liposome encapsulated [Fe(CN)6]3- through channels was fundamental for the change of NPs' sizes. The optimized PB NP-liposome composite showed higher adsorption capacity of Cs+ ions than that of aggregated PB NPs that are prepared without liposome in aqueous media.

Sensing of fluoride ions in aqueous media using a luminescent coordination polymer and liposome composite.

The synthesis of a luminescent coordination polymer (Tb-BTC) within the confined environment of a liposome allowed for the anisotropic growth of nanosized Tb-BTC crystals. Consequently, the resulting composite Tb-BTC@Lipo exhibited a higher fluorescence sensitivity to fluoride anions in aqueous media compared to the independent Tb-BTC in bulk.

Lipophilic ruthenium salen complexes: incorporation into liposome bilayers and photoinduced release of nitric oxide.

A new lipophilic Ru salen complex with cholesterol groups can be efficiently incorporated into liposome bilayers, allowing the photoinduced release of nitric oxide (NO) and the membrane transport of NO to coexisting liposomes.

Regulation of a cerium(IV)-driven O2 evolution reaction using composites of liposome and lipophilic ruthenium complexes.

A composite containing a liposome and a lipophilic ruthenium complex was synthesized to regulate an O2 evolution reaction using cerium(IV) ammonium nitrate as an oxidizing reagent. We found that the surrounding environment of the reaction centre is an important factor for controlling the O2 evolution catalytic reaction. We successfully regulated the reaction activity using the linker length of the lipophilic ligand and using the head groups of the phospholipid component.

Guest responsivity of a two-dimensional coordination polymer incorporating a cholesterol-based co-ligand.

To implement specific guest responsivity, a hydrophobic cholesterol-based co-ligand, cholest-5-en-3-yl-4-isonicotinate (Cholpy), was incorporated into a two-dimensional Hofmann-type Co(II)Ni(II) coordination polymer. The chemically programmed structure successfully demonstrated the unique guest response with remarkable chromatic changes.

Dual modification of a triple-stranded ß-helix nanotube with Ru and Re metal complexes to promote photocatalytic reduction of CO2.

We have constructed a robust ß-helical nanotube from the component proteins of bacteriophage T4 and modified this nanotube with Ru(II)(bpy)(3) and Re(I)(bpy)(CO)(3)Cl complexes. The photocatalytic system arranged on the tube catalyzes the reduction of CO(2) with higher reactivity than that of the mixture of the monomeric forms.

Elucidation of metal-ion accumulation induced by hydrogen bonds on protein surfaces by using porous lysozyme crystals containing Rh(III) ions as the model surfaces.

Metal-ion accumulation on protein surfaces is a crucial step in the initiation of small-metal clusters and the formation of inorganic materials in nature. This event is expected to control the nucleation, growth, and position of the materials. There remain many unknowns, as to how proteins affect the initial process at the atomic level, although multistep assembly processes of the materials formation by both native and model systems have been clarified at the macroscopic level. Herein the cooperative effects of amino acids and hydrogen bonds promoting metal accumulation reactions are clarified by using porous hen egg white lysozyme (HEWL) crystals containing Rh(III) ions, as model protein surfaces for the reactions. The experimental results reveal noteworthy implications for initiation of metal accumulation, which involve highly cooperative dynamics of amino acids and hydrogen bonds: i) Disruption of hydrogen bonds can induce conformational changes of amino-acid residues to capture Rh(III) ions. ii) Water molecules pre-organized by hydrogen bonds can stabilize Rh(III) coordination as aqua ligands. iii) Water molecules participating in hydrogen bonds with amino-acid residues can be replaced by Rh(III) ions to form polynuclear structures with the residues. iv) Rh(III) aqua complexes are retained on amino-acid residues through stabilizing hydrogen bonds even at low pH (approximately 2). These metal-protein interactions including hydrogen bonds may promote native metal accumulation reactions and also may be useful in the preparation of new inorganic materials that incorporate proteins.

Modification of porous protein crystals in development of biohybrid materials.

Protein assemblies have attracted increasing attention for construction of biohybrid materials. Protein crystals can also be regarded as solid protein assemblies. The present work demonstrates that protein crystals can be employed as porous biomaterials by site-specific modifications of the crystals of recombinant sperm whale myoglobin mutants. The myoglobin crystals of space group P6 provide hexagonal pores consisting of the building blocks of six Mb molecules, which form a pore with a diameter of 40 A. On the basis of the lattice structure of the Mb crystals, we have selected appropriate residues located on the surface of the pores for replacement with cysteine. This enables modification of the pore surface via coupling with maleimide derivatives. We have succeeded in crystallizing the modified Mb mutants, retaining the P6 lattice, and consistently aligning nanosized functional molecules such as fluorescein, eosin, and Ru(bpy)(3) into the hexagonal pores of the Mb crystals. Our strategy for site-specific modification of protein crystal pores is applicable to various protein crystals with porous structures. We believe that modified porous protein crystals will provide attractive candidates for novel solid materials in nanotechnology applications.

Construction of an energy transfer system in the bio-nanocup space by heteromeric assembly of gp27 and gp5 proteins isolated from bacteriophage T4.

Protein assemblies, such as viruses and ferritins, have been employed as useful molecular templates for the accumulation of organic and inorganic compounds to construct bio-nanomaterials. While several methods for conjugation of heterofunctional molecules with protein assemblies have been reported, it remains difficult to control their fixation sites in the assemblies. In this article, we demonstrate the three-dimensional arrangement of different types of fluorescent probes using the heteromeric self-assembly of (gp27-gp5)(3) which is the component protein of bacteriophage T4 (gp: gene product). The composites exhibited fluorescence resonance energy transfer from fluorescein to tetramethylrhodamine dyes immobilized in the bio-nanocup space. The alternation of the donor and acceptor positions induced fluorescence self-quenching by the formation of ground-state complexes of the acceptors. These results indicate that the site-specific conjugation method using the bio-nanocup space of the heteromeric protein assembly has potential for the integration of several types of functional molecules in protein nanospaces.

Coordinated design of cofactor and active site structures in development of new protein catalysts.

New methods for the synthesis of artificial metalloenzymes are important for the construction of novel biocatalysts and biomaterials. Recently, we reported new methodology for the synthesis of artificial metalloenzymes by reconstituting apo-myoglobin with metal complexes (Ohashi, M. et al., Angew Chem., Int. Ed. 2003, 42, 1005-1008). However, it has been difficult to improve their reactivity, since their crystal structures were not available. In this article, we report the crystal structures of M(III)(Schiff base).apo-A71GMbs (M = Cr and Mn). The structures suggest that the position of the metal complex in apo-Mb is regulated by (i) noncovalent interaction between the ligand and surrounding peptides and (ii) the ligation of the metal ion to proximal histidine (His93). In addition, it is proposed that specific interactions of Ile107 with 3- and 3'-substituent groups on the salen ligand control the location of the Schiff base ligand in the active site. On the basis of these results, we have successfully controlled the enantioselectivity in the sulfoxidation of thioanisole by changing the size of substituents at the 3 and 3' positions. This is the first example of an enantioselective enzymatic reaction regulated by the design of metal complex in the protein active site.