Differences in Lipid Order and Dynamics in Plasma Membranes Assessed by Nonlinear Optical Microscopy.

When amphiphilic polar dyes were added to the cells, they intercalated predominantly in the outer leaf of the plasma membrane, making them active for second harmonic generation (SHG). The fluorescence of the dye enabled simultaneous 3D imaging of SHG and two-photon excited fluorescence (TPF). Because SHG intensity is sensitive to the alignment of the dyes, which reflects lipid ordering in the plasma membrane, we assessed the difference in lipid ordering by comparing the SHG intensity normalized to the TPF intensity. Together with an enzyme release assay that detects pore formation in the plasma membrane, our SHG assay revealed how polycations affect lipid ordering at low concentrations, where membrane damage has not yet been examined. By scaling the results of the assays with the charge concentration of the two polycations, polyethylenimine (PEI) and poly-l-lysine (PLL), we found that PEI reduced the lipid order more than PLL, and PLL formed more pores than PEI. A comparison of the SHG and TPF images of the wounded cells revealed that one of the lipid dynamics (flip-flop) was significantly enhanced in the bleb membrane. Moreover, the SHG assay indicated that the biocompatible polymer, poly(N-(2-hydroxypropyl)methacrylamide), did not affect the lipid order. Thus, our technique allows the assessment of the plasma membrane structure at the molecular level.

Optical second harmonic generation microscopy: application to the sensitive detection of cell membrane damage.

Optical second harmonic generation (SHG) is a nonlinear optical process which is sensitive to the symmetry of media. SHG microscopy allows for selective probing of a non-centrosymmetric area of sample. This type of nonlinear optical microscope was first used to observe ferroelectric domains and has been applied to various specimens including the biological samples to date. Imaging of the endogenous SHG of biological tissue has been utilized for the selective observation of filament systems in tissues such as collagen, myosin, and microtubules, which exhibit a polar structure. The cellular membrane can be selectively observed by the SHG microscope through membrane staining with amphiphilic polar dye molecules. It has been reported that, by imaging exogenous SHG of the membrane, sensitive detection of membrane damage could be realized using the SHG microscope. Because the staining dye is fluorescent, both SHG and two-photon excited fluorescence (TPF) images can be obtained simultaneously. How the SHG intensity depends on the molecular alignment of the polar dye molecules that reflects the ordering of lipid molecules in the plasma membrane and the necessity of the normalization of the SHG intensity by the TPF intensity is discussed. Furthermore, the assessment of the membrane damage induced by exposing polycation to HeLa cells has been compared with the conventional cytotoxicity and cell viability tests to demonstrate the higher sensitivity of the present SHG-based assay.

Partially induced transition from horizontal to vertical orientation of helical peptides at the air-water interface and the structure of their monolayers transferred on the solid substrates.

To apply the Langmuir-Blodgett (LB) technique as a platform for investigating the fundamental properties of amphiphilic peptides (APs), we have investigated the structure of LB films using the APs. To vertically orient the helical APs like transmembrane proteins in the membrane, the primary structure of the APs was designed to have two domains: a hydrophilic domain (three amino acids) and a hydrophobic domain (ca. 20 amino acids). However, we are still far from having full control of their orientation. This study reports the contribution of the subphase temperature to the change in the orientation of helical APs. When the surface pressure-area isotherm of AP was observed at the subphase temperature at 41.5°C, the isotherm exhibited a plateau, implying that a phase transition of the monolayer at the air-water interface occurred. Circular dichroism (CD) spectra of the monolayers transferred on the solid substrates revealed that the orientation of the helices changed at the pressure, where the plateau of the isotherm was observed. This change was not observed at 21.5°C, i.e., the horizontal alignment of helixes was maintained. Atomic force microscopy (AFM) was used to systematically investigate the surface structure of the monolayers transferred at different surface pressures. A structural model of the monolayer that did not contradict with the results obtained by the three different techniques (the isotherm, CD spectroscopy, and AFM) was derived, and it was concluded that the horizontally oriented helices partially changed their orientation to vertical upon compression in the plateau region of the isotherm.

Fabrication of Langmuir-Blodgett films using amphiphilic peptides.

To introduce self-organization ability of transmembrane proteins into Langmuir (L) and Langmuir-Blodgett (LB) techniques, we focused on "amphiphilic peptide" (AP) which is composed of two distinct hydrophilic and hydrophobic domains. Three types of APs of different average hydropathies were used to prepare the AP/lipid mixed L and LB films. According to the circular dichroism spectra, the secondary structures of APs were not uniform but were a mixture of alpha-helix, beta-strand and random coil. The fraction of alpha-helix was higher for lower hydropathy AP. The interaction between AP and lipid in the L film and the structure of the LB film were also depended on the APs used.

Characteristics of halorhodopsin-bacterioruberin complex from Natronomonas pharaonis membrane in the solubilized system.

Halorhodopsin is a retinal protein with a seven-transmembrane helix and acts as an inward light-driven Cl(-) pump. In this study, structural state of the solubilized halorhodopsin (NpHR) from the biomembrane of mutant strain KM-1 of Natronomonas pharaonis in nonionic detergent was investigated. A gel filtration chromatography monitored absorbances at 280 and 504 nm corresponding to the protein and a lipid soluble pigment of bacterioruberin (BR), respectively, has clearly detected an oligomer formation of the NpHRs and a complex formation between the NpHR and BR in the solubilized system. A molar ratio of NpHR:BR in the solubilized complex was close to 1:1. Further SDS-PAGE analysis of the solubilized NpHR cross-linked by 1% glutaraldehyde has revealed that the NpHR forms homotrimer in detergent system. Although this trimeric structure was stable in the presence of NaCl, it was dissociated to the monomer by the heat treatment at 45 °C in the desalted condition. The same tendency has been reported in the case of trimeric NpHR expressed heterologously on the E. coli membrane, leading to a conclusion that the change of strength of the trimeric association dependent on the ion binding is a universal feature of the NpHR. Interestingly, the trimer dissociation on the NpHR was accompanied by the complete dissociation of the BR molecule from the protein, indicated that the cavity formed by the NpHR protomers in the trimeric conformation is important for tight binding of the BR. Because the binding affinity for Cl(-) and the resistance to hydroxylamine under light illumination showed only minor differences between the NpHR in the solubilized state and that on the biomembrane, the influences of solubilization to the tertiary structure and function of the protein are thought to be minor. This NpHR-BR complex in the solubilized system has a potential to be a good model system to investigate the intermolecular interaction between the membrane protein and lipid.

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl ß-D-maltoside.

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl ß-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.

Synthesis of monodisperse mesoporous silica hollow microcapsules and their release of loaded materials.

Monodisperse mesoporous silica hollow capsules (MSHCs) with sizes ranging from 570 to 75 nm were synthesized using the sol-gel method combined with the template-assisted method. Monodisperse polystyrene (PS) particles were used as templates for the hollow structure of the MSHCs, and cylindrical micelles of cationic surfactant were used to create mesopores across the shell of the MSHC. To obtain MSHCs with a degree of polydispersity in diameter comparable to that of the PS templates and spherical in shape with uniform shell thicknesses, the conditions for the synthesis were systematically examined. It was found that the ranges of the reaction conditions to obtain such MSHCs had to be narrow because (1) the colloidal stability of the particles must be maintained before and after the sol-gel reaction and (2) the rate of the silica formation during the reaction must be regulated to attain sufficient shell thickness to retain the hollow structure and to achieve smooth surfaces. The sustained release of dye molecules loaded in the MSHCs was confirmed, indicating that our MSHC is a candidate for use as a drug carrier in drug delivery systems or as a container for microreactors.

Determination of a merocyanine J-aggregate structure and the significant contribution of the electric dipole interaction to the exciton band wavelength.

The molecular alignment of a merocyanine (MC) J-aggregate monolayer at the air-water interface was determined by a grazing incidence x-ray diffraction method. The obtained molecular arrangement apparently shows that the conventional formula, which accounts only for the transition dipole interaction, is not sufficient to figure out the exciton band wavelength, suggesting the importance of the electric dipole (ED) interaction. We derived a simple formula for the ED interaction energy under an extended dipole approximation and clarified the ED contribution in the MC J aggregate.

Homogeneous, competitive fluorescence quenching immunoassay based on gold nanoparticle/polyelectrolyte coated latex particles.

This study reports a homogeneous and competitive fluorescence quenching immunoassay based on gold nanoparticle/polyelectrolyte (Au(NP)/PE) coated latex particles prepared by the layer-by-layer (LbL) technique. First, the resonant energy transfer from a layer of fluorescent PEs to Au(NP) in LbL assembled films on planar substrates was investigated. The quenching efficiency (QE) for the planar films depended on the cube of the distance between the two layers. A QE of 50% was achieved at a distance of ca. 15 nm, indicating that the Au(NP)/PE system is suitable for detecting binding/release events for antibodies. A homogeneous, competitive binding immunoassay for biotin was designed based on Au(NP)/PE-coated polystyrene particles of 488 nm diameter as quenching agents for a fluorescein isothiocyanate labeled anti-biotin immunoglobulin (FITC-anti-biotin IgG). Biotin molecules were localized on the Au(NP)/PE-coated latexes by depositing a layer of biotinylated poly(allylamine hydrochloride) (B-PAH), and FITC-anti-biotin IgGs were subsequently bound to the particles through interaction with the biotin on B-PAH. Transmission electron microscopy and quartz crystal microgravimetry confirmed the multilayer formation on latex particles and planar gold surfaces, respectively. The biotin-functionalized Au(NP)/PE-coated latexes terminated by FITC-anti-biotin IgG exhibited a dynamic sensing range of 1-50 nmol. These results indicate that Au(NP)/PE-coated latexes can be readily used as dynamic range tunable sensors.

Preparation of J-aggregate liposome dispersions and their chromic transformation.

We report the preparation of aqueous liposome dispersions of J-aggregates formed by the amphiphilic merocyanine dye (MD). A series of liposome-forming lipids were dispersed together with MD J-aggregates at different molar ratios of MD to lipid. The MD J-aggregate dispersions prepared with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at the MD to DMPC ratio of 0.16 exhibit good dispersibility; that is, they can be readily redispersed without any flocculation even after their precipitation. By use of different counterions for the MD molecules, two types of J-aggregate dispersions, one that exhibits an absorption band (J-band) at 635 nm (type I) and the other at 600 nm (type II), were obtained. As an example of the use of MD J-aggregates liposome dispersions, the thermochromic transformation of MD J-aggregates was demonstrated. When the dispersions are heated, J-aggregates of type I transformed into type II at a certain temperature (T(disp)). The parameters that control the speed of the transformation and the value of T(disp) were determined.