Sadato Yabuki

Fine Structure in the Thermal Denaturation of DNA: High Temperature-Resolution Spectrophotometric Studie

Fine structures which appear in the optical melting profile of DNA are examined from both the experimental and theoretical aspects. After a brief historical survey of the DNA melting experiments during the pre-fine-structure era in Section II, the high temperature-resolution experimental techniques which are essential to the investigation of fine structure are described in Section III. Then, the current status of the high-resolution study is reviewed first by a phenomenological description of the melting profile (Section IV) and then of the refolding profile (Section V), where a general idea about the cooperatively melting region and several factors affecting it is given. Sections VI and VII are devoted to the review of current theoretical works. Several well-established theoretical frameworks which correlate the base sequence with the melting phenomena are examined in terms of their rigorousness and usefulness. The molecular thermodynamic parameters concerning the DNA melting which have been evaluated by several research groups are compared and discussed. Finally, in Section VIII, current ideas on the correlation between the fine structure and genetic functions and genetic maps are reviewed. Some future problems relating to the fine structure are also discussed.

Structure and reactivity of aerosol-OT reversed micelles containing α-chymotrypsin

The structure and reactivity of α-chymotrypsin solubilized in biological buffer–sodium bis(2-ethylhexyl)sulfosuccinate (AOT)–2,2,4-trimethylpentane (isooctane) reversed micelles, have been studied at various water/surfactant molar ratios (ωo) and protein concentrations using synchrotron radiation X-ray small-angle scattering and enzymatic activity measurements. Two types of α-chymotrypsins and two different substrates (p-nitrophenyl acetate and p-nitrophenyl caprylate) were used. The scattering experiments show that α-chymotrypsins are entrapped in the water pools. In the low ωo range (0 < ωo < 12) oligomeric AOT reversed micellar formation occurs as a metastable state and simultaneously the enzymatic activity is enhanced significantly for both types of α-chymotrypsins, showing a maximum activity at ωo≈ 12 for both substrates. The results from the scattering and enzymatic activity measurements suggest that the hydrolysis of the above esters, catalysed by α-chymotrypsins, is enhanced at the internal interface of the AOT reversed micelle and that the metastable oligomeric phase plays an important role in accelerating the metabolic turnover by increasing the apparent interfacial area of the micelles close to the enzymes.

Fine structures in denaturation curves of bacteriophage lambda DNA. Their relation to the intramolecular heterogeneity in base composition

Precise recording of polyphasic optical melting curves was carried out for three kinds of bacteriophage lambda DNA differing in length (lambdac1857s7, lambdacIb2 and lambdacIb2b5). Each of denaturation steps in melting profiles was characterized by two parameters, the melting temperature and the relative size. Any difference in fine structures in melting profiles was not recognized between the intact lambdacI857s7DNA and the DNA fragmented into halves. The change in fine structures in melting profiles caused by the deletions of the b2 and b5 region agreed qualitatively well with the prediction based on the physical and the genetical maps of phage lambda chromosome. The combined results indicate that, first, the well-known linear relationship between melting temperature and G+C content may apply also to each of denaturation steps in polyphasic melting curves due to heterogeneity of nucleotide distribution in a single DNA species, and, second, the effect of molecular ends on melting fine structures can be neglected at moderate salt concentration (0.01 M less than or equal to Na+ less than or equal to 0.2 M) for such a high molecular weight DNA. The heterogeneous distribution of nucleotides was derived for lambdaDNA and for its b2 and b5 regions.

Thermotropic phase behavior and stability of monosialoganglioside micelles in aqueous solution

The thermotropic phase behavior of monosialoganglioside in a dilute aqueous dispersion at pH 6.8 was measured by using synchrotron radiation small-angle x-ray scattering and was analyzed by a shell-modeling method. Previous calorimetric studies on ganglioside systems have shown quite different thermotropic behaviors from other biological lipid systems, however, the details have still been ambiguous. Because of high statistical data and a shell-modeling analysis, we could elucidate the internal structural change of monosialoganglioside micelle induced by the elevation of temperature from 6 to 60 degrees C, that is, the shrinkage of the hydrophilic region and the slight expansion of the hydrophobic region occurring simultaneously, accompanying the elongation of the axial ratios of the ellipsoidal micelles. The model structures obtained explain the changes in the experimental scattering curves, the distance distribution functions, and the gyration radii. In addition we have also found an evident thermal hysteresis in the scattering curves and in the structural parameters. The present result suggests that the thickness of the hydrophilic region, namely, the conformation of oligosaccharide chains, is sensitive to a change of temperature.

INTERMICELLAR INTERACTION OF GANGLIOSIDE AGGREGATES AND STRUCTURAL STABILITY ON PH VARIATION

We have studied the effect of concentration and pH variations on micellar structures of sialoglycosphingolipids (gangliosides) by using synchrotron radiation small-angle X-ray scattering. We have treated four different aqueous dispersions of gangliosides containing monosialogangliosides (GM1), disialogangliosides (GD1a) and two different types of the ganglioside mixtures. To discuss the change in the scattering curve with concentration in the range of 0.1–10% w/v, we employed a rescaled mean spherical approximation method (RMSA) for charged particle dispersions combined with a shell-modelling method. The present modelling method well describes the characteristics of the whole experimental scattering curve, namely an intermicellar correlation peak at a very small angle and a rounded peak at a medium angle reflecting an intramicellar scattering density fluctuation. We determined simultaneously the inter- and intra-micellar structures of the ganglioside aggregates under the repulsive screened Coulomb potential between them. The estimated micellar surface charge is ca. 57 e in 0.01 M citrate buffer at pH 6.7, 25 °C. For every sample, the pH elevation from 3.6 to 8.0 at 25 °C caused similar changes in the experimental scattering curve, gyration radius and distance distribution function, suggesting a shrinkage of the micellar dimension and an intramicellar structural change. The modelling analyses can explain the above changes mostly resulting from the change of the oligosaccharide chain portions in the micelles. The structural differences depending on the specimens, containing different ganglioside components are also clarified.

Microcalorimetric studies of Ca2+ binding to triphosphoinositide and phosphatidylserine

Abstract The effects of Ca 2+ ions on the physicochemical properties of an aqueous dispersion of phosphatidylinositol-4,5-bis(phosphate) (TPI) were investigated by microcalorimetry in comparison with the effects of Ca 2+ ions on the properties of glycerylphosphoryl-inositoldiphosphate (GPIDP) and phosphatidylserine (PS). The mixing enthalpy per mole of the aqueous dispersion of TPI with the CaCl 2 solution was always positive in the whole range of CaCl 2 concentration, while those of GPIDP and PS were negative. A model to explain this unusual result is presented. The hydrocarbon chains of TPI molecules contribute positively to the mixing enthalpy, while those of PS make a negative contribution. The positive contribution of the hydrocarbon chains of TPI to the mixing enthalpy is attributed to the dehydration enthalpy of the hydrophobic hydration induced by Ca 2+ ions. In the absence of Ca 2+ ions, some spaces are likely to be produced between their hydrocarbon chains because of strong electrostatic repulsion between the large negative charges of the head groups of TPI and also because of the loose packing due to the highly unsaturated chain of the arachidonic acid of TPI. Then, the hydrophobic hydration in the interior of the TPI membrane would be made possible. The influence of pH on the mixing enthalpy of the aqueous dispersion of TPI with the CaCl 2 solution also agrees perfectly with this model.

Characteristics of thermotropic phase transition of glycosphingolipid (Ganglioside) aggregates in aqueous solution

Abstract In this report, by using synchrotron radiation X-ray small-angle scattering, we show the structural characteristics of the ganglioside aggregates depending on both the temperature and oligosaccharide chain. The experimental results of the aqueous solutions containing G M1 , G D1 , or crude mixture gangliosides show that the elevation of temperature induces the change of the micellar structure accompanied by the change of the internal scattering density distribution. These changes are well described by applying a shell-modeling method to the observed scattering curves since the shell-modeling method can determine the intramicellar structures. A noticeable common feature found in the thermotropic transition of the ganglioside micelles is the contraction of the micellar dimension resulting from the shrinkage of the hydrophilic region of the micelle. The shrinkage amounts to 20–30% of the thickness of the hydrophilic region, suggesting the structural change of the oligosaccharide chains of the gangliosides from an extended conformation to a compact one. The differences of the thermotropic stability depending on the number of the sialic acids of the oligosaccharide chain were also observed. The present results which suggest a thermal perturbation in the physiological range 20–50°C induces a conformational change of the oligosaccharide chains of ganglioside molecules very sensitively.

Salt-dependent phase behaviour of the phosphatidylinositol 4,5-diphosphate–water system

We have studied the effect of mono- and di-valent cations on the structure of the phosphatidylinositol 4,5-diphosphate (PIP2)–biological buffer system at pH 7.2. We found that the PIP2 aggregates from prolate ellipsoidal micelles at pH 7.2 and that the addition of a small amount of Ca2+ ions even below 5 mmol dm–3([Ca2+] : [PIP2]= 5/7) induces a drastic structural phase transition from prolate micelles to disordered lamellae. Mg2+ ions induced a similar phase transition around 10 mmol dm–3, which, however, occurred less sensitively. In contrast to the case of divalent cations, the presence of Na+ ions did not affect the PIP2 micellar structures up to 0.1 mol dm–3([Na+] : [PIP2]= 100/7), indicating that the above structural change of the PIP2 aggregates is not attributable to the elevation of the electrostatic shielding effect between the polar heads. Modelling analysis suggests that the observed phase transition results from an increase of the critical packing parameter of the PIP2 molecule accompanying the dehydration of the PIP2 polar-head region.

Complexes of gangliosides with proteins in solution

The interaction of gangliosides with methylated bovine albumins was studied using neutron and synchrotron-radiation X-ray solution scattering methods. The present result suggests that the binding of gangliosides with protein occurs heterogeneously.

Calorimetric and small angle X-ray and neutron scattering studies of mixing gangliosides and methylated albumin

We measured the mixing enthalpies of gangliosides with methylated albumin in aqueous solutions at various concentration ratios, and carried out X-ray, neutron scattering experiments for the mixed solutions at nearly the same concentration ratios. The mixing heats were positive when the mixing ratios of lipid to protein inclined to either side, and negative at the intermediate range of the mixing ratio corresponding to the formation of interfacial fluff in a binary solution reported by Hayashi. The X-ray and neutron scattering analyses of the mixed solutions showed that addition of methylated albumin induces disintegration of the micelle structure of gangliosides and patchlike binding of ganglioside molecules to the albumin molecule appears. The analyses also showed that origomeric particles of the lipoproteins are formed at the intermediate range of the mixing ratios.