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

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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.