INTERMEDIATES AND THE BARRIERS IN PROTEIN FOLDING

在目前的赠款期间开展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。此外，还进行了动力学实验以确定 细胞色素c在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。拟议中的工作 旨在测试和构建这些假设，这些假设可以列出 具体如下。1)展开状态：在正常溶液条件下， 未折叠多肽以非特异性收缩链的形式存在。在……里面 从稀释展开开始的停流折叠实验 蛋白质来自高变性剂，这种偏向的U条件是非常达到的 快速(不到1毫秒)，并不代表高效折叠 中级的。2)成核：折叠开始于初始 大力上山进行大规模构象搜索以达到 可以支持前向折叠步骤的过渡状态核 以下坡的方式，因此在某些拓扑结构上必须是原生的 有道理。这种基于扩散搜索的步骤通常需要1到 10毫秒。3)折叠中间体：来自折叠核的细胞 C链通过三个连续的序列能量下山 越来越像本地的、协作式结构的中间体，以 原生状态。从一个中间体到下一个中间体的步骤只需要 一种快速、小规模的构象搜索。4)错误障碍：在此 下坡序列中的复性蛋白可能具有较高的 概率，遇到一个或多个可选的、依赖于错误的错误折叠- 重组障碍可能会减缓原生州的收购， 通常(对于小的蛋白质)到大约1秒的时间尺度。 这些假设将通过应用我们早先的氢气来验证 交换脉冲标记法，我们更新的本征态氢交换 方法。2D核磁共振、其他光谱(CD、荧光、吸光度)、 快速反应方法(停流法)。这些实验将使用 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。

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.