INTERMEDIATES AND THE BARRIERS IN PROTEIN FOLDING

蛋白质折叠的中间体和障碍

• 批准号: 2838487
• 负责人: S. Walter ENGLANDER
• 金额: $ 33.26万
• 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/2838487
• 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.

当前赠款期间的工作开发了新的氢交换 似乎在细胞色素c中定义了三个中间体的方法 折叠路径。 还进行了动力学实验以定义 细胞色素C必须克服其折叠轨迹的障碍。 获得的信息指向一组连贯的步骤 描述细胞色素c折叠的主要途径。拟议的工作 旨在测试和基于这些假设，可以列出 如下。 1）展开状态：在正常溶液条件下， 展开的多肽作为非特殊收缩链存在。 在 停止流量折叠实验，从稀释开始 来自高变性剂的蛋白质，这种有偏见的U条件非常到达 迅速（小于1毫秒），并不代表生产性折叠 中间的。 2）成核：折叠始于和初始 大规模上坡大规模构象搜索以达到 过渡状态核可以支持在 下坡的方式，因此必须像某种拓扑结构一样像本地 感觉。 这个扩散搜索依赖性步骤通常需要1至 10毫秒。 3）折叠中间体：从折叠核，Cyt C链通过三个序列进行能量下坡移动 越来越类似于本地的，合作的结构化中间体 本地国家。 从一个中间到另一个中间的步骤仅需要 快速，小规模的构象搜索。 4）错误障碍：在此 下坡序列重折叠蛋白可能会以高 概率，遇到一个或多个可选的，错误依赖的错误折叠 - 重组障碍可以减缓本地国家的收购， 通常（对于小蛋白质）至大约1秒的时间尺度。 这些假设将通过应用我们的早期氢来检验 交换脉冲标签方法，我们的新天然状态氢交换 方法。 2D NMR，其他光谱（CD，荧光，吸光度）， 和快速反应方法（停止流）。 实验将使用 哺乳动物，细菌和基因设计的细胞色素版本 C作为模型蛋白和其他有用的模型蛋白。