Teruaki Onai

Collapse of the hydration shell of a protein prior to thermal unfolding

Based on high statistical quality wide-angle X-ray scattering data for the unfolding–refolding process of hen egg-white lysozyme (HEWL), we have analysed the change of the hydration shell as a function temperature using the program CRYSOL. The present results suggest that the decrease of the hydration-shell density starts from a lower temperature than the transition temperature of the collapse of the tertiary structure of HEWL. Although the use of CRYSOL for scattering data for proteins before the transition has an apparent limitation, the collapse of the hydration shell prior to the unfolding of HEWL agrees with a slight tendency of the radius of gyration to decrease during the thermal unfolding process.

Morphology transition of raft-model membrane induced by osmotic pressure: Formation of double-layered vesicle similar to an endo- and/or exocytosis

The effect of osmotic pressure on the structure of large uni-lamellar vesicle (LUV) of the lipid mixtures of monosialoganglioside (GM1)-cholesterol-dioleoyl-phosphatidylcholine (DOPC) was studies by using wide-angle X-ray scattering (WAXS) method. The molar ratios of the mixtures were 0.1/0.1/1, 0/0.1/1, and 0/0/1. The ternary lipid mixture is a model of lipid rafts. The value of osmotic pressure was varied from 0 to 4.16×105 N/m2 by adding the polyvinylpyrrolidone (PVP) in the range from 0 to 25 % w/v. In the case of the mixtures without GM1, the rise of the osmotic pressure just enhances the multi-lamellar stacking with deceasing the inter-lamellar spacing. On the other hand, the mixture containing GM1 shows the structural transition from a uni-lamellar vesicle to a double-layered vesicle (a liposome including a smaller one inside) by the rise of osmotic pressure. In this morphology transition the total surface area of the double-layered vesicle is mostly as same as that of the LUV at the initial state. The polar head region of GM1 is bulky and highly hydrophilic due to the oligosaccharide chain containing a sialic acid residue. Then, the present results suggest that the existence of GM1 in the outer-leaflet of the LUV is essentially important for such a double-layered vesicle formation. Alternatively, a phenomenon similar to an endo- and/or exocytosis in cells can be caused simply by a variation of osmotic pressure.

Initial process of amyloid formation of apomyoglobin and effect of glycosphingolipid GM1

Using small-angle and wide-angle X-ray scattering techniques it has been possible to clarify the helix-to-sheet (cross-β) transition and the stacking process of the cross-β sheet of apomyoglobin as a model for amyloid. The present results indicate that the cross-β formation and the pleated sheet stacking start concurrently and that the stacking continues progressively after the saturation of the cross-β formation. The effect of glycosphingolipids on the above processes has also been studied. At high molar ratio of glycosphingolipid to apomyoglobin the growth of the amyloid is suppressed.

Effect of osmotic pressure on ganglioside-cholesterol-DOPC lipid mixture

By means of small-angle X-ray scattering (SAXS) method, we have studied the structure of the lipid mixtures of monosialoganglioside (GMI)-cholesterol-dioleoyl-phosphatidylcholine (DOPC) system as a model of lipid raft. The samples were small uni-lamellar vesicle (SUV) except for GMI sample. The osmotic pressure was changed with varying the polyvinylpyrrolidone (PVP) concentration in the range from 0 to 25 % w/w. The increase of the PVP concentration is known to reduce the lamellar spacing due to the increase of the osmotic pressure. On the other hand the polar head region of GMI was shown to be highly hydrophilic by the presence of oligosaccharide chain containing one sialic acid residue. In the cases of the GMI micelle and GMI-cholesterol SUV the presence of PVP affects little on those aggregate structures. In the case of the SUVs of cholesterol-DOPC the stacking of the bilayers was induced with the increase of PVP concentration, especially at high cholesterol content. In the case of the SUVs of GMI-cholesterol-DOPC the multi-lamellar stacking was suppressed, but a minor change of the SUV structure was induced. The present results suggest that the coexistence of GMI and cholesterol affords the lipid bilayer a resistance to the osmotic stress and avoids a multi-layered stacking.

Permeability of water through a raft model membrane clarified by time-resolved SANS and SAXS

We have characterized the structures of ganglioside (GD3) micelle and GD3/cholesterol mixtures, and have determined the permeability of water across a membrane of GD3/cholesterol/phospholipid uni-lamellar vesicle. We have found that monovalent Na+ and K+ cations affect the permeability of water differently. We have found also that the permeability of water is greatly enhanced by the K+ ion, suggesting the function of the GD3/cholesterol rich clusters on neuronal excitations by K+. The complimentary use of small-angle X-ray and neutron scattering techniques is useful to determine the permeability of water across a vesicle membrane.