FOLDING AND CHEMICAL MODIFICATION OF RIBONUCLEASE A

核糖核酸酶 A 的折叠和化学修饰

• 批准号: 3438866
• 负责人: ROGER G BIRINGER
• 金额: $ 9.46万
• 依托单位: SAN JOSE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3438866
• 关键词: chemical stability; chemical structure function; conformation; enzyme structure; fluorescence spectrometry; isomer; nuclear magnetic resonance spectroscopy; pancreatic ribonuclease; peptide chemical synthesis; protein folding; protein structure; thermodynamics

An understanding of how proteins fold is of major biological importance. This proposal is addressed to the factors which control the folding pathway and thereby define the native structure. The long-term goals of this project are to determine how the amino acid sequence guides the folding process. Knowledge of the "code" which determines the way in which proteins fold would be an invaluable tool for a number of disciplines. In light of the recent advances in genetic engineering, one could, in principle, design a protein with a particular structure and function or redesign a protein in order to make it more stable than its horizon s for the use and production of proteins for medical research and pharmaceuticals. The equilibrium unfolding/refolding and the kinetics of refolding of bovine ribonuclease A derivatives will be studied using absorbance, fluorescence, and high-field 1H nuclear magnetic resonance (NMR). A detailed analysis of intermediate structures along the folding path will be made. Both thermal and chaotropic agent induced structures will be examined. Initial experiments will involve the preparation and characterization of chemically modified derivatives of bovine pancreatic ribonuclease A. In particular, nitrotyrosyl and aminotryrosyl derivatives will be examined. In later experiments the modified residues will serve as structural probes for various portions of the protein molecule and facilitate the assignments of resonances in the 1H NMR spectrum. The unfolding and refolding transitions will be followed and the changes in the signals derived from the probes, along with the known amino acid sequence and native crystal structure, employed to determine changes in structure. Several different sets of unfolding conditions will be examined to determine their effect of the unfolded state structure and the pathway of folding. The project will utilize aqueous-methanol solvent systems to facilitate stabilization of partially-folded protein conformations. This approach is unique and has been quite successful in previous studies.

了解蛋白质如何折叠具有重要的生物学意义。 这一建议是针对控制折叠途径的因素 从而限定原生结构。 长期目标是 项目是确定氨基酸序列如何指导折叠 过程 了解决定蛋白质折叠方式的“密码” 将是许多学科的宝贵工具。 考虑到 在基因工程的最新进展，人们可以，原则上，设计一个 或重新设计蛋白质， 为了使它比地平线更稳定， 用于医学研究和制药。 牛血清白蛋白的去折叠/复性平衡及复性动力学 核糖核酸酶A衍生物将使用吸光度，荧光， 和高场1H核磁共振（NMR）。 详细分析 将制作沿着折叠路径的中间结构。 两个热 和离液剂诱导的结构。 初步实验将涉及制备和表征 牛胰腺核糖核酸酶A的化学修饰衍生物。 在 特别是硝基酪氨酸和氨基酪氨酸衍生物。 在以后的实验中，修饰的残基将作为结构探针 蛋白质分子的不同部分，并促进分配 1H NMR谱中的共振。 展开和折叠 转换将被遵循，并且信号中的变化从 探针，沿着已知的氨基酸序列和天然晶体 结构，用于确定结构的变化。 几种不同 一组展开条件将被检查，以确定它们的效果， 展开状态结构和折叠路径。 该项目将利用含水甲醇溶剂系统， 稳定部分折叠的蛋白质构象。 这种方法 它是独一无二的，在以前的研究中非常成功。