Hydrogen exchange, proteins, and folding

氢交换、蛋白质和折叠

基本信息

批准号：
6430117
负责人：
S. Walter ENGLANDER
金额：
$ 52.67万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-12-01 至 2005-11-30
项目状态：
已结题

项目摘要

Work in the present period studied the determinants of protein hydrogen exchange (HX) and employed HX capabilities and other methods to study global and subglobal stability, structure change, and protein folding. Systems studied were cytochrome c (Cyt c), ribonuclease A, a hyperthermophilic rubredoxin, the GroE system, and whole active muscle. Some major advances were made. A model proposed for the protein folding process provides a coherent explanation for available information on the equilibrium and the kinetic steps in protein folding in terms of a classical more or less sequential pathway of distinct intermediates and defined barriers, proceeding within the developing native-like context. A new 'stability labeling' method to test for the on-pathway nature of intermediates was successfully applied to the folding intermediates previously found in Cyt c. A facile high yield recombinant Cyt c system was produced and used to generate mutants designed to explore the folding process. This system will also be useful for a manifold of other Cyt c studies. A forceful energy-dependent unfolding of a substrate protein by the GroE system was demonstrated. Other goals were accomplished. Initiatives proposed will build on these advances. A histidine scanning series will be used to kinetically trap and populate the different intermediates in Cyt c folding; the intermediates can then be separately identified and their structural and kinetic relationships established. A glycine scanning series will be used in the equilibrium stability labeling mode to test for the on-pathway nature of Cyt c folding intermediates and to help establish this approach for other proteins more generally. The glycine mutants will also be used to characterize the previously enigmatic 'local fluctuations' that determine the HX behavior of most protein hydrogens under most conditions. An approach will be attempted that promises to make the native state HX method generally applicable to the majority of proteins which have so far not been accessible to this analysis. Other work is planned.

目前的工作研究了蛋白质氢交换（HX）的决定因素，并利用HX能力和其他方法来研究全局和亚全局稳定性、结构变化和蛋白质折叠。研究的系统包括细胞色素 c (Cyt c)、核糖核酸酶 A、超嗜热红氧还蛋白、GroE 系统和整个活动肌肉。取得了一些重大进展。为蛋白质折叠过程提出的模型为蛋白质折叠中的平衡和动力学步骤的可用信息提供了连贯的解释，根据不同中间体和定义障碍的经典或多或少顺序途径，在发展中的类似天然的背景下进行。一种新的“稳定性标记”方法可以测试中间体的通路性质，该方法已成功应用于先前在 Cyt c 中发现的折叠中间体。生产了一种简便的高产重组 Cyt c 系统，并用于生成旨在探索折叠过程的突变体。该系统也可用于多种其他 Cyt c 研究。证明了 GroE 系统对底物蛋白的强力能量依赖性解折叠。其他目标都实现了。拟议的举措将建立在这些进步的基础上。组氨酸扫描系列将用于动力学捕获和填充 Cyt c 折叠中的不同中间体；然后可以分别识别中间体并建立它们的结构和动力学关系。甘氨酸扫描系列将用于平衡稳定性标记模式，以测试 Cyt c 折叠中间体的通路性质，并帮助更普遍地为其他蛋白质建立这种方法。甘氨酸突变体还将用于表征以前神秘的“局部波动”，这些波动决定了大多数蛋白质氢在大多数条件下的 HX 行为。我们将尝试一种方法，使天然状态 HX 方法普遍适用于迄今为止无法进行此分析的大多数蛋白质。其他工作正在计划中。