Masakatsu Hato

Self-assembling properties of synthetic peptidic lipids

Novel peptidic lipids were synthesized by the coupling of a linear oligopeptide as the hydrophilic moiety with a glutamic acid dialkylamide as the hydrophobic moiety. Their self-assembling properties were investigated. The critical aggregate concentrations (CAC) for the peptidic lipids with a double dodecyl group were in the range of 1.0 x 10(-5)-3.8 x 10(-5) M. The phase transition parameters and the aggregation morphologies in aqueous dispersion were largely dependent on the number and nature of the constitutive amino acid residues. Dark-field optical microscopy demonstrated that the present peptidic lipids can form four types of stable morphologies in water, i.e., tubular structures, twisted ribbons, vesicles, and amorphous crystals.

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids.

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.

Hydration and molecular motions in synthetic phytanyl-chained glycolipid vesicle membranes.

Proton permeation rates across membranes of a synthetic branch-chained glycolipid, 1,3-di-O-phytanyl-2-O-(beta-D-maltotriosyl)glycerol (Mal3(Phyt)2) as well as a branch-chained phospholipid, diphytanoylphosphatidylcholine (DPhPC) were lower than those of straight-chained lipids such as egg yolk phosphatidylcholine (EPC) by a factor of approximately 4 at pH 7.0 and 25 degrees C. To examine whether degrees of water penetration and molecular motions in Mal3(Phyt)2 membranes can account for the lower permeability, nanosecond time-resolved fluorescence spectroscopy was applied to various membranes of branch-chained lipids (Mal3(Phyt)2, DPhPC, and a tetraether lipid from an extremely thermoacidophilic archaeon Thermoplasma acidophilum), as well as straight-chained lipids (EPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and digalactosyldiacylglycerol (DGDG)) using several fluorescent lipids. Degrees of hydration of glycolipids, Mal3(Phyt)2, and DGDG were lower than those of phospholipids, EPC, POPC, and DPhPC at the membrane-water interfaces. DPhPC showed the highest hydration among the lipids examined. Meanwhile, rotational and lateral diffusive motions of the fluorescent phospholipid in branch-chained lipid membranes were more restricted than those in straight-chained ones. The results suggest that the restricted motion of chain segments rather than the lower hydration accounts for the lower proton permeability of branch-chained lipid membranes.

Regio- and stereocontrolled synthesis of d-erythro-sphingosine and phytosphingosine from d-glucosamine

d-erythro-Sphingosine (1) and phytosphingosine (2) have been efficiently synthesized from d-glucosamine by utilizing its whole carbon skeleton and functional groups. In this synthetic route, regioselective alkylation of the epoxy-tosylate 9 was achieved with a copper(I)-catalyzed Grignard reagent to give the key intermediate 10, which was converted to both 1 and 2 via regioselective formation of the iodohydrin 11.

Synthesis of 1,3-di-O-alkyl-2-O-(β-glycosyl)glycerols bearing oligosaccharides as hydrophilic groups

A novel series of synthetic glycolipids, 1,3-di-O-alkyl-2-O-(beta-glycosyl)glycerols, and their efficient synthetic route were proposed. These glyceroglycolipids were synthesized in good overall yields and stereoselectivity in five steps via trichloroacetimidate glycosylation with 1,3-di-O-alkylglycerols. This route was applied to prepare the glycolipids bearing a cello- or malto-oligosaccharide with a definite number of sugar residues from one to six. Thin-layer chromatography, elemental analysis, nuclear magnetic resonance spectroscopy and infrared absorption spectroscopy confirmed that these glycolipids were chemically pure compounds.

Polymer-dispersed bicontinuous cubic glycolipid nanoparticles.

We found that certain amphiphilic polymers such as PEO‐PPO‐PEO triblock copolymer (PL) can directly disperse a cubic glycolipid, 1‐O‐phytanyl‐β‐d‐xyloside (β‐XP), into bicontinuous cubic nanoparticles in water medium. The use of synchrotron small‐angle X‐ray diffraction (SSAXD) permitted the identification of the exact structure of these dispersed particles in the colloidal state. Dynamic light scattering method was used to obtain particle size distributions. The dispersion quality and the dispersion time can be improved by co‐dissolving the lipid and the polymer in a common solvent. The mean volume diameter of these dispersed colloidal particles depends on the mixing time and polymer concentration. About 5 wt % (0.18 mol %) of polymer to lipid weight was found to be sufficient to produce stable colloidal dispersions. At this polymer content and at 3 h of stirring time, the mean volume diameter of cubic colloidal particles was found to be 1.0 μm. Increase of dispersion time to 6 h reduced the colloidal particle size from 1.0 μm to 660 nm. At 3 h of mixing time, the increase of polymer content, from ∼5 to ∼10 wt %, reduced the particle mean diameter from 1.0 μm to 675 nm. Irrespective of these dispersion times and polymer contents, the dispersed colloidal particles exhibit predominately the Pn3m cubic phase structure, the same as that of a β‐XP‐water binary mixture, although a weak coexistence of Im3m cubic phase is identified in these colloidal particles. This coexistence is found to have the characteristics of a Bonnet relation, which forms convincing evidence for the infinite periodic minimal surface descriptions (IPMS). Considering the biotechnological significance, the preparation of these colloidal dispersions was carried out in a phosphate‐buffered saline (PBS) system. These cubic colloidal dispersions exhibited good stability and the cubic phase structure remained intact in the PBS system.

Glycolipid-cholesterol monolayers: towards a better understanding of the interaction between the membrane components.

In this work, the interaction between a synthetic analog of archaeal lipids and cholesterol was studied using Langmuir technique. The lipid, β-Mal(3)O(C(16+4))(2), contained phytanyl chains attached via two ether bonds to the sn-2 carbon of the glycerol backbone. The preliminary studies showed that monolayers formed with the pure lipid have a liquid-like character; here, a hypothesis that admixing cholesterol to β-Mal(3)O(C(16+4))(2) could confer a higher rigidity on the films was tested. To check this proposal, two-dimensional miscibility of cholesterol and β-Mal(3)O(C(16+4))(2) in monomolecular films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy and polarization-modulation infrared reflection absorption spectroscopy. The stability of the monomolecular films was evaluated based on thermodynamics of mixing of cholesterol and β-Mal(3)O(C(16+4))(2). Atomic level information concerning the orientation of molecules and the degree of hydration of polar headgroups was obtained from molecular dynamics simulations.

Colloidal properties of aqueous bivalent metal dodecylpoly(oxyethylene)sulfates and hexadecylpoly(oxyethylene)sulfates (I)

Abstract Effects of alkyl chain length, oxyethylene chain length, and various gegenions on the colloidal properties of surfactant type CmH2m+1(OC2H4)nSO41/2M have been studied. The results are summarized as follows. (1) Krafft points of CmH2m+1(OC2H4)nSO41/2M (m = 12, 16) are depressed by an increase in oxyethylene chain length (n); therefore this surfactant type can be used in hard water as well as in the presence of heavy metal ions. (2) Foam drainage and creaming of O/W emulsion (benzene—0.5 wt% aqueous surfactant, 1:1 in volume) are remarkably retarded by an increase in alkyl chain length of the calcium salts from 12 to 16, while those properties of the aqueous sodium salts are not so affected. (3) A viscoelasticity develops in the dilute micellar solutions of bivalent metal alkylpoly(oxyethylene)sulfates the alkyl chain length of which is longer than 16, e.g., C16H33SO41/2M (M = Cu, Mn, Ca), C16H33(OC2H4)nSO41/2M, and C18H37SO41/2Cu. The viscosity of the micellar solutions of these surfactants is high and non-Newtonian. On the other hand, the viscoelasticity does not develop in the micellar solutions of bivalent metal salts of lower homologs and sodium salts [CmH2m+1(OC2H4)nSO4Na (m = 12, 16; n = 1, 2, 3) and CmH2m+1SO4Na (m = 12, 14, 16, 18)] over the concentration range up to 5–10 wt%. A possible mechanism of the characteristics of the aqueous C16H33(OC2H4)nSO41/2M solutions has been discussed.

Surface forces between protein A adsorbed mica surfaces

Abstract The effects of protein concentration, pH and ionic strength ( I ) of aqueous electrolytes on the forces between recombinant protein A (rPA) adsorbed mica surfaces were studied. All force-distance curves can be described in terms of two different regimes, a “longer distance” regime and an “adsorbed layer” regime. In the “longer distance” regime, the forces are largely due to an electrostatic double-layer force originating from the charges on the adsorbed rPA layers. For the forces between rPA layers adsorbed from 100 ppm at pH 3.5, this interpretation was confirmed by the good agreement between the surface potential ψ 0 inferred from the force curve, and the ζ-potential of rPA adsorbed mica surfaces. In the “adsorbed layer” regime, forces of steric origin dominate owing to the overlap of adsorbed layers. From the analysis of the forces in the “adsorbed layer” regime, possible conformations of the adsorbed rPA molecules were estimated. In solutions of low ionic strength ( I =1 mM, pH 3.5–8.1) and at low rPA concentrations, rPA molecules are adsorbed “side-on” parallel to the surfaces forming a monolayer. With increasing rPA concentrations, rPA molecules appear to form a “pseudo-double-layer”. Both the adsorbed layer thickness (“hard wall” thickness) and the adsorbed amount do not change appreciably with pH. When the ionic strength of the solution is raised ( I ≈ 100 mM), the “adsorbed layer” regime expands to about 25–30 nm from the mica surface. This is due to more “extended” conformations of adsorbed rPA molecules.

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3

The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.