INTERMEDIATES AND THE BARRIERS IN PROTEIN FOLDING

蛋白质折叠的中间体和障碍

• 批准号: 6125271
• 负责人: S. Walter ENGLANDER
• 金额: $ 34.01万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6125271
• 关键词: analytical chemistry; chemical kinetics; conformation; cytochrome c; intermolecular interaction; molecular chaperones; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; stop flow technique

Work in the present grant period developed a new hydrogen exchange method that appears to define three intermediates in the cytochrome c folding pathway. Also kinetic experiments were done to define the barriers that cytochrome c must overcome in their folding trajectory. The information obtained points to a coherent set of steps that may describe the major pathway in cytochrome c folding. The proposed work is designed to test and build on these hypotheses, which can be listed as follows. 1) The unfolded state: Under normal solution conditions, unfolded polypeptides exist as non-specifically contracted chains. In stopped-flow folding experiments that start by diluting unfolded proteins from high denaturant, this biased U condition is reached very rapidly (less than 1 msec), and does not represent a productive folding intermediate. 2) Nucleation: Folding begins with and initial energetically uphill large scale conformational search to reach a transition state nucleus that can support forward folding steps in a downhill manner, and therefore must be native-like in some topological sense. This diffusional search-dependent step typically requires 1 to 10 msec. 3) Folding intermediates: From the folding nucleus, the cyt c chain moves energetically downhill through a sequence of three increasingly native-like, cooperatively structured intermediates to the native state. The step from one intermediate to the next requires only a fast, small scale conformational search. 4) Error barriers: In this downhill sequence the refolding protein may, often with high probability, encounter one or more optional, error-dependent misfold- reorganization barriers that can slow native state acquisition, typically (for small proteins) to the approximate 1 second time scale. These hypotheses will be tested by applying our earlier hydrogen exchange pulse labeling method, our newer native state hydrogen exchange method. 2D NMR, other spectroscopies (CD, fluorescence, absorbance), and rapid reaction methods (stopped-flow). The experiments will use mammalian, bacterial, and genetically engineered versions of cytochrome c as model proteins, and other useful model proteins.

当前资助期内的工作开发了一种新的氢交换 该方法似乎定义了细胞色素 c 中的三个中间体 折叠路径。 还进行了动力学实验来定义 细胞色素 c 在其折叠轨迹中必须克服的障碍。 获得的信息指向一组连贯的步骤，这些步骤可以 描述细胞色素 c 折叠的主要途径。拟议的工作 旨在测试和建立这些假设，这些假设可以列出 如下。 1）展开状态：在正常溶液条件下， 未折叠的多肽以非特异性收缩链的形式存在。 在 从稀释展开开始的停流折叠实验 来自高变性剂的蛋白质，这种偏向的 U 条件非常容易达到 快速（小于 1 毫秒），并不代表有效的折叠 中间的。 2）成核：折叠开始于 和 初始 大力开展大规模构象搜索以达到 过渡态核可以支持向前折叠步骤 下坡方式，因此在某些拓扑中必须是类似本地的 感觉。 这种依赖于扩散搜索的步骤通常需要 1 到 10 毫秒。 3）折叠中间体：来自折叠核，细胞色素 c 链通过三个序列有力地向下移动 越来越像原生的、协作结构的中间体 原生状态。 从一个中间步骤到下一个中​​间步骤只需要 快速、小规模的构象搜索。 4）错误障碍：在此 重折叠蛋白的下坡序列可能通常具有高 概率，遇到一个或多个可选的、与错误相关的错误折叠- 重组障碍可能会减缓本土国家的收购， 通常（对于小蛋白质）大约为 1 秒的时间尺度。 这些假设将通过应用我们早期的氢来检验 交换脉冲标记方法，我们较新的本态氢交换 方法。 2D NMR、其他光谱（CD、荧光、吸光度）、 和快速反应方法（停流）。 实验将使用 哺乳动物、细菌和基因工程版本的细胞色素 c 作为模型蛋白，以及其他有用的模型蛋白。