PATHWAYS OF PROTEIN FOLDING

蛋白质折叠途径

• 批准号: 3484321
• 负责人: ROBERT L BALDWIN
• 金额: $ 25.86万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-01-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3484321
• 关键词: DNA; acidity /alkalinity; bacteria; bacterial proteins; chemical kinetics; chemical reaction; conformation; cytochrome c; enzyme structure; genetic manipulation; isozymes; nuclear magnetic resonance spectroscopy; nucleoproteins; pancreatic ribonuclease; protein engineering; protein folding; protein sequence; stop flow technique; trypsin inhibitors

Our work is focussed on determining the pathway and understanding the mechanism of protein folding. We are engaged in two main projects. The first is determination of the kinetic pathway of folding of ribonuclease A. There are at least 3 forms of the unfolded protein, caused perhaps by the cis-trans isomerization of proline residues after unfolding, and we study the refolding pathway of the major slow-folding form. In previous work we have shown that structural intermediates are well-populated transiently during folding at 0 degree 10 degrees C, and that numerous peptide NH protons are protected against exchange with H2O before the tertiary structure is formed. By a D20 trapping method, followed by 2D 1H-NMR after folding is complete, we are determining the structural locations of the protected peptide NH protons. In future work we will use pulse exchange experiments to measure the degree of protection of each protected proton as a function of time during folding. The second main project is to find out if apomyoglobin is a suitable system for testing the framework model of folding and to investigate the role of the helix:helix pairing sites during folding. Privalov has found a prominent equilibrium intermediate in the folding of sperm whale apomyoglobin, whose properties resemble the so-called "molten globule state". We find that human apomyoglobin does not show this intermediate although dog and horse myoglobin do show it, and we propose to use site-directed mutagenesis to find out which amino acid residues are responsible. We will also use the "medium-resolution method" of Rosa and Richards (1979) to find out which alpha-helices are present as judged by protected peptide NH protons.

我们的工作专注于确定道路和理解 蛋白质折叠的机制。 我们参与了两个主要 项目。 首先是确定的动力学途径 核糖核酸酶的折叠A。至少有3种形式 未折叠的蛋白质，可能是由顺式传播异构化引起的 展开后脯氨酸残留物，我们研究重折叠 主要缓慢折叠形式的途径。 在以前的工作中，我们有 表明结构中间体瞬时占 在0度10度C折叠过程中，并且众多肽 NH质子受到保护，免受与H2O的交换 三级结构形成。 通过D20捕获方法，遵循 折叠完成后2d 1H-NMR，我们正在确定 受保护肽NH质子的结构位置。 在 未来的工作我们将使用脉冲交换实验来测量 每个受保护的质子随时间的函数的保护程度 在折叠过程中。 第二个主要项目是找出apomyoglobin是否是 适合测试折叠框架模型的系统 研究螺旋的作用：螺旋配对位点 折叠式的。 Privalov发现了一个突出的平衡中间体 在抹香鲸的折叠中，其特性 类似于所谓的“熔融球体”。 我们发现人类 apomyoglobin尽管狗和马不显示这种中间体 Myoglobin确实显示了它，我们建议使用以网站为导向的 诱变以找出哪些氨基酸残基是造成的。 我们还将使用Rosa的“中分辨率方法”和 理查兹（Richards，1979）找出存在哪些α-螺旋。 由受保护的肽NH质子判断。