HIGH PRESSURE NMR STUDIES OF PROTEINS

拟议研究的长期目标是促进 了解其稳定性、折叠中间体和动力学 以压力作为基本热力学变量。 独特的高压设备，适用于0.1兆帕以下的测量(L 巴)到L的平均绩点(10千巴)和从零下25摄氏度到零下摄氏度，将是 与各种L-D和二维高分辨核磁共振配合使用 研究压力对展开/折叠的影响的技术 平衡，自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， 这些蛋白质的冷变性行为将通过采取 利用压力降低水的凝固点的优点。 模型的压力、冷、热和化学变性状态 将对蛋白质进行检查和比较，特别强调 冷变性和加压变性观察到致密的变性状态。这个 为这些研究选择的模型蛋白质包括蛋清溶菌酶， 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、葡萄球菌突变体的筛选 核酸酶和牛胰腺核糖核酸酶A。 以补充在高压下的核磁共振测量，特别是在 模型蛋白及其突变体的致密变性状态 二向色性(CD)和激光化学诱导的动态核 将进行偏振(CIDNP)实验。高压CD 细胞和CIDNP探针将被设计和建造。

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.