Solution X-ray scattering study on protein structure at high pressure using synchrotron

本研究旨在研制高压溶液X射线散射仪，并将其应用于蛋白质溶液体系。在研制工作中，通过改进支承环的设计和压力产生机理，组成了高性能的高压仪（最大500 MPa）。在应用工作中，我们测量了压力下溶菌酶和肌红蛋白的溶液X射线散射（SOXS）。为了估计蛋白质的可压缩性，我们测定了溶菌酶在压力下的回转半径（Rg），在此半径范围内蛋白质保持天然结构。溶菌酶的Rg随压力的增加而减小：1atm时为14.85 *，300 MPa时为14.46 *。这意味着Rg的变化为-0.13 */100 MPa。该值的绝对值明显大于-0.04 */100 MPa。为了阐明蛋白质的压力去折叠的特性，我们测量了肌红蛋白在PH 4.4和压力高达30 MPa下的SOXS。Rg与压力的关系呈S形曲线，在300 MPa时达到最大值。结果表明，中点压力约为200 MPa，在300 MPa时蛋白质变性最好。在1atm和300 MPa下的Rg值分别为17.5 * 和21.5 *。在300 MPa下的值明显小于肌红蛋白的变性剂解折叠所报道的值。压力解折叠的值与熔融球态的压力解折叠的值相当接近。

The purposes of this study are to develop the high-pressure solution X-ray scattering instrument and to apply the technique to the protein solution system. In the development work, we have composed the high-perfomance high-pressure instrument (max.500 MPa) by improving the design of the backup ring and the mechanics of pressure generation. In the application work, we have measured the solution X-ray scattering (SOXS) from lysozyme and myoglobin under pressure. For the estimation of compressibility of protein, we determined the radius of gyration (Rg) of lysozyme under pressure, up to which the protein keeps the native structure. The Rg of lysozyme decreases with increasing pressure : 14.85 * at 1 atm, 14.46 * at 300 MPa. It means that the change in Rg is -0.13 */100 MPa. This value in the absolute is remarkably larger than -0.04 */100 MPa. To clarify the characteristic of pressure unfolding of protein, we measured SOXS from myoglobin at PH 4.4 under pressure up to 30 MPa. The pressure dependence of Rg showed the sigmoid curve reaching to the maximum at 300 MPa. It indicated that the midpoint pressure is about 200MPa, and that the protein prefectly denatured at 300 MPa. The values of Rg at 1atm and 300 MPa are 17.5 * and 21.5 *, respectively. The value at 300 MPa is remarkably smaller than the values pound 30 * reported for the denaturant unfolding of myoglobin. The value for pressure unfolding is rather close to that for molten globule state.