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

本研究的目的是研制高压溶液x射线散射仪，并将该技术应用于蛋白质溶液体系。在研制工作中，我们研制了高性能高压仪器（max500）通过改进备份环的设计和压力产生的机理。在应用工作中，我们测量了溶菌酶和肌红蛋白在压力下的溶液x射线散射（SOXS）。为了估计蛋白质的可压缩性，我们确定了溶菌酶在压力下的旋转半径（Rg），在此半径内蛋白质保持天然结构。溶菌酶的Rg随压力的增加而降低，1atm时为14.85 *，300mpa时为14.46 *。即Rg的变化量为-0.13 */ 100mpa。该绝对值明显大于-0.04 */ 100mpa。为了阐明蛋白质的压力展开特性，我们测量了PH 4.4下肌红蛋白在压力高达30 MPa下的SOXS。Rg的压力依赖性在300 MPa时达到最大，呈s型曲线。结果表明，中点压力约为200MPa，蛋白质在300mpa时变性最明显。1atm和300 MPa时的Rg值分别为17.5 *和21.5 *。300兆帕时的值明显小于30 *磅报道的肌红蛋白变性展开值。压力展开的值与熔球状态的值相当接近。

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.