PROTEIN FOLDING AND NMR AND MUTAGENESIS

这个项目的目的是了解折叠的机制， 一些代表性的模型蛋白质的结构和动力学术语。 的 蛋白质折叠问题至今仍是生物学中未解决的主要问题之一。 分子生物学，尽管其基本的重要性和实用性， 对蛋白质工程和生物技术的影响。 最近 二维核磁共振氢交换方法的进展 我们实验室开发的标记和快速混合技术使其 可以观察到氢键结构的形成，在不同的 再折叠的阶段，从而确定结构的顺序， 折叠途径上的中间体。 为了阐明 在折叠中的特定相互作用，这些结构工具将被连接起来， 具有通过定点突变进行有意的氨基酸改变的能力， 诱变 氢交换脉冲标记和竞争方法将用于 完成褶皱构造描述的初步工作 野生型泛素的中间体。 这些信息将提供 框架进一步分析的折叠机制，由网站导向 诱变 一种有效的诱变和细菌表达系统， 基于合成的遍在蛋白基因，是可用的。 突变将被 旨在改变折叠过程中的关键相互作用， 蛋白质的稳定性和折叠动力学将通过光谱分析来监测。 方法和1D NMR。 有限数量的突变体将被选择用于更多 详细的脉冲标记和2D NMR研究。 类似的实验策略将应用于以下平行研究： 硫氧还蛋白的野生型和突变形式。 主要的缓慢折叠阶段 这种蛋白质将被消除的手段，延长脉冲标记 蛋白质仅短暂暴露于解折叠的方案 条件 在更大的阶段，我们将把这项工作扩展到泛素融合蛋白， 为了阐明泛素在核糖体中可能的伴侣功能， 组装和蛋白质表达。 泛素-肌红蛋白和泛素- 将构建硫氧还蛋白融合蛋白并在E.杆菌 在初始光谱表征之后，我们将应用脉冲 标记和2D NMR方法进行更详细的研究， 泛素连接对硫氧还蛋白折叠行为的影响。

This project is aimed at understanding the mechanism of folding in structural and kinetic terms for some representative model proteins. The protein folding problem remains today one of the major unsolved problems in molecular biology, despite its fundamental importance and practical implications with respect to protein engineering and biotechnology. Recent advances in two-dimensional NMR methods together with hydrogen exchange labeling and rapid mixing techniques developed in our laboratory make it possible to observe the formation of H-bonded structure at the various stages of refolding and thus to define the sequence of structural intermediates on the folding pathway. In order to elucidate the role of specific interactions in folding, these structural tools will be joined with the ability to make deliberate amino acid changes by site-directed mutagenesis. Hydrogen exchange pulse labeling and competition methods will be used to complete preliminary work on the structural description of folding intermediates in wild-type ubiquitin. This information will provide the framework for further analysis of the folding mechanism by site-directed mutagenesis. An efficient mutagenesis and bacterial expression system, based on a synthetic ubiquitin gene, is available. Mutations will be designed to alter key interactions in folding, and the resulting changes in protein stability and folding kinetics will be monitored by spectroscopic methods and 1D NMR. A limited number of mutants will be selected for more detailed pulse labeling and 2D NMR studies. Similar experimental strategies will be applied in parallel studies on wild-type and mutant forms of thioredoxin. The dominant slow folding phase of this protein will be eliminated by means of an extended pulse labeling scheme in which the protein is only briefly exposed to unfolding conditions. At a larger stage we will extend this work to ubiquitin fusion proteins in order to address the possible chaperon function of ubiquitin in ribosomal assembly and protein expression. Ubiquitin-myoglobin and ubiquitin- thioredoxin fusion proteins will be constructed and expressed in E. coli. After an initial spectroscopic characterization we will apply pulse labeling and 2D NMR methods for a more detailed study of the effect of ubiquitin attachment on the folding behavior of thioredoxin.