Shigeki Arai

Reversibility and hierarchy of thermal transition of hen egg-white lysozyme studied by small-angle x-ray scattering.

To clarify mechanisms of folding and unfolding of proteins, many studies of thermal denaturation of proteins have been carried out at low protein concentrations because in many cases thermal denaturation accompanies a great tendency of aggregation. As small-angle x-ray scattering (SAXS) measurements are liable to use low-concentration solutions of proteins to avoid aggregation, SAXS has been regarded as very difficult to observe detailed features of thermal structural transitions such as intramolecular structural changes. By using synchrotron radiation SAXS, we have found that the presence of repulsive interparticle interaction between proteins can maintain solute particles separately to prevent further aggregation in thermal denaturation processes and that under such conditions the thermal structural transition of hen egg-white lysozyme (HEWL) holds high reversibility even at 5% w/v HEWL below pH approximately 5. Because of the use of the high concentration of the solutions, the scattering data has enough high-statistical accuracy to discuss the thermal structural transition depending on the structural hierarchy. Thus, the tertiary structural change of HEWL starts from mostly the onset temperature determined by the differential scanning calorimetry measurement, which accompanies a large heat absorption, whereas the intramolecular structural change, corresponding to the interdomain correlation and polypeptide chain arrangement, starts much prior to the above main transition. The present finding of the reversible thermal structural transitions at the high protein concentration is expected to enable us to analyze multiplicity of folding and unfolding processes of proteins in thermal structural transitions.

Complementary Analysis of Thermal Transition Multiplicity of Hen Egg-White Lysozyme at Low pH Using X-ray Scattering and Scanning Calorimetry

In this present paper we present a new method of small-angle X-ray scattering (SAXS) data analysis (called the TMA method) to examine multiplicity of structural transition of denaturation processes of proteins. The TMA method gives us a molar fraction of spatial-conformational-state and an indication of deviation from the two-state structural transition hypothesis in a structural transition process. Using this method we have successfully compared a spatial-conformational-state transition observed by SAXS with a thermodynamicmicrostate transition observed by differential scanning calorimetry (DSC) in the thermal denaturation process of hen egg-white lysozyme (HEWL) at the pH range of 2.4-3.1. The TMA method is able to dissect spatialconformational-state transition multiplicity depending on structural hierarchy. We have found that the thermal structural transition of HEWL is well-characterized mostly in terms of two different spatial-conformationalstate transitions in the tertiary and intramolecular structures. Upon heating, the transition-midpoint temperature of the spatial-conformational-state transition of the tertiary structure mostly agrees with that of DSC, while the transition-midpoint temperature of the intramolecular structure greatly differs from that of DSC. The tertiary structural transition essentially obeys the two-state transition feature with a sharp critical temperature in comparison with the intramolecular structural transition. When the pH is lowered from 3.1 to 2.4, the former transition tends to shows a maximum deviation from the two-state transition feature around the transition temperature. On the other hand, the intramolecular structural transition proceeds continuously with a relatively large deviation in the whole temperature range measured at all pH values. The present results obtained by the TMA method essentially agree with our previous report using standard SAXS analyses in HEWL system at different pH range of 7.0-1.2, and would afford us further aspects to discuss experimental evidences with theoretical models of protein folding, such as nucleation-collapse models.

Interaction of gangliosides with proteins depending on oligosaccharide chain and protein surface modification.

By using neutron and synchrotron x-ray small-angle scattering techniques, we investigated the process of the complexation of gangliosides with proteins. We treated monosialoganglioside (G(M1)), disialoganglioside (G(D1a)), and a mixture of G(M1)/G(D1a). Proteins used were bovine serum albumins whose surfaces were modified with different sugars (deoxy-D-galactose, deoxy-L-fucose, deoxymaltitol, and deoxycellobiitol), which were used as model glycoproteins in a membrane. We found that the complexation of gangliosides with albumins greatly depends on the combination of ganglioside species and protein surface modification. With a varying protein/ganglioside ratio in a buffer solution at pH 7, the complexation of G(M1) or G(D1a) with albumins modified by monosaccharides appears to be less destructive for ganglioside aggregate structures in forming large complexes; the complexation of G(D1a) with the albumins modified by disaccharides induces the formation of complexes with a dimeric structure; and the complexation of G(M1) with albumins modified by disaccharides, to form small complexes, is very destructive. The present results show a strong dependence of the interaction between ganglioside and protein on the characteristics of the ganglioside and protein surface, which would relate to a physiological function of gangliosides, such as a function regulating the receptor activity of glycoproteins in a cell membrane.

Dynamics of w/o AOT microemulsions studied by neutron spin echo

Abstract By using neutron spin echo and synchrotron radiation small-angle X-ray scattering, we have studied the structure and dynamics of water/sodium bis(2-ethylhexyl)sulfosuccinate/d 16 - n -heptane microemulsion with varying water/surfactant molar ratio, w 0 . We have found the anomalous q -dependence of the effective diffusion coefficient D eff at low w 0 whose peak-position at low w 0 does not satisfy the predicted relation of qR = π ( R , microemulsion radius). This would relate to the presence of the oligomeric phase at low w 0 observed by SAXS.

Concentration dependence of thermal structural transition of hen egg-white lysozyme under constant heating rate studied by time-resolved SAXS

Differential scanning calorimetry (DSC) measurements are well known to serve us heat capacity functions of macromolecules and molar fractions of thermodynamic microstates. On the other hand, small-angle X-ray scattering (SAXS) measurements are expected to have an advantage for determining directly spatial-conformational states of macromolecules since we can observe ensemble-averaged scattering functions from solute macromolecules at multiple spatial-conformational states. In the present paper we will present an approach to analyze spatial-conformational-state transitions observed in denaturation processes of proteins by SAXS, which affords us a new aspect of thermal transition of proteins in comparison with thermodynamic-microstate transitions observed by DSC. From the point of view of spatial-conformational-state transition, we will clarify the thermal structural transition aspects of hen egg-white lysozyme (HEWL) at pH 5 depending on the conformational hierarchy and concentration.

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.

Comparison of DMSO-induced denaturation of hen egg-white lysozyme and bovine α-lactalbumin

Abstract By using both wide-angle neutron and small-angle X-ray scattering techniques, we have studied the denaturation processes of hen egg-white lysozyme (HEWL) and bovine α-lactalbumin (BLA) in dimethyl sulfoxide (DMSO)/water mixtures. Although these proteins are known to be highly homologous, the present results indicate that the structure of BLA is liable to be destroyed by the addition of DMSO in comparison with the case of HEWL. DMSO-induced denaturation is suggested to relate to the collapse of hydration shell surrounding the protein surface.

Solvent contrast variation study of w/o AOT microemulsions using small-angle neutron scattering

Abstract Recently, we showed that the hydrolysis of some esters catalyzed by α-chymotrypsin entrapped in sodium bis(2-ethylhexyl)sulfosuccinate (AOT) microemulsions is enhanced at low water-surfactant molar ratio w 0 ( = [H 2 O]/[AOT]) and that the internal interface structure and the oligomeric phase of the AOT microemulsions below this w 0 range would play an important role for the acceleration of the metabolic turnover. To clarify the relation between the appearance of superactivity and the internal structures of the AOT microemulsions, we have carried out small-angle neutron-scattering experiments. In this report, we treat the AOT/water/hexane or heptane system by using solvent-contrast variation method, and we discuss a change of the excluded volume of AOT molecule at low w 0 value.

Wide-angle neutron scattering study of thermal denaturation of lysozyme

Abstract Many studies of folding-unfolding transition of protein have been carried out for the clarification of the fundamental biological problem of protein folding, namely, how a polypeptide chain attains its native conformation. Although one of the approaches to solve protein-folding mechanisms is evidently to follow denaturation processes of protein, there is little direct evidence showing intramolecular structural changes under thermal perturbation. By using a wide-angle neutron-scattering spectrometer, we have monitored the thermal denaturation process of hen egg-white lysozyme at different pH under the unaggregative condition. The present results show that the temperature elevation gradually induces an intramolecular structural fluctuation prior to a main transition of the tertiary structure without accompanying significant heat absorption, and the multiplicity in the thermal transition observed suggests the presence of hierarchic stability depending on the structural hierarchy which would characterize an energetic landscape of folding-unfolding transition of protein.