HIGH PRESSURE NMR STUDIES OF PROTEINS

拟议研究的长期目标是促进 了解的稳定性，折叠中间体，和动力学的 用压力作为基本热力学变量来计算蛋白质。 独特的高压设备，适用于从0.1 MPa（l） 巴）至lGPa（10千巴）以及从-25摄氏度至100摄氏度，将是 与各种1-D和2-D高分辨率NMR结合使用 研究压力对展开/折叠影响的技术 平衡，天然和致密变性状态的构象状态， 以及几种模型蛋白质的动力学性质。 此外该 这些蛋白质的冷变性行为将被研究， 水的凝固点通过压力降低的优点。 模型的压力、冷、热和化学变性状态 蛋白质将被检查和比较，特别强调 通过冷变性和压力变性观察到的致密变性状态。的 选择用于这些研究的模型蛋白包括鸡蛋白溶菌酶， 噬菌体T4溶菌酶，马肌红蛋白，抹香鲸肌红蛋白， 葡萄球菌核酸酶和牛胰腺核糖核酸酶A，以及 T4溶菌酶、抹香鲸肌红蛋白、葡萄球菌 核酸酶和牛胰腺核糖核酸酶A。 为了补充高压下的NMR测量，特别是在 模型蛋白及其突变体的紧凑变性状态，环状 二向色性（CD）和激光光化学诱导动态核 极化（CIDNP）实验。 高压CD 细胞和CIDNP探针将被设计和构建。

The long-term objective of the proposed research is to contribute to the understanding of the stability, folding intermediates, and dynamics of proteins by using pressure as the fundamental thermodynamic variable. Unique high pressure equipment, suitable for measurements from 0.1 MPa (l bar) to l GPa (10 kbar) and from -25 degrees C to lOO degrees C, will be used in conjunction with a variety of l-D and 2-D high resolution NMR techniques to study the effects of pressure on the unfolding/folding equilibria, conformational states of native and compact denatured states, and the dynamic properties of several model proteins. In addition, the cold-denaturation behavior of these proteins will be studied by taking advantage of the depression of the freezing point of water by pressure. The pressure, cold, heat, and chemically denatured states of the model proteins will be examined and compared, with special emphasis on the compact denatured states observed by cold and pressure denaturation. The model proteins selected for these studies include hen egg-white lysozyme, bacteriophage T4 lysozyme, horse myoglobin, sperm-whale myoglobin, staphylococcal nuclease, and bovine pancreatic ribonuclease A, as well as selected mutants of T4 lysozyme, sperm-whale myoglobin, staphylococcal nuclease, and bovine pancreatic ribonuclease A. To complement the NMR measurements at high pressure, especially on the compact denatured states of the model proteins and their mutants, circular dichroism (CD) and laser-photo chemically induced dynamic nuclear polarization (CIDNP) experiments will be performed. High-pressure CD cells and CIDNP probes will be designed and constructed.