FOLDING OF THE ACID STATE, SUBDOMAINS AND INTACT RNASE H

这项提议的目标是了解蛋白质的氨基酸序列如何决定其折叠和稳定性。为了实现这一目标，我们需要了解给定序列可获得的所有构象的结构和稳定性，即它的能量景观。这方面的困难在于蛋白质是非常合作的。为了绕过这个问题，我们将蛋白质的结构和稳定性解剖成更小的片段，并研究了蛋白大肠杆菌核糖核酸酶H的部分折叠构象和折叠部分。通过比较动力学和平衡实验的结果，我们获得了令人惊讶的结论：所有部分折叠的构象(酸性熔融球体、本征状态的高能构象和动力学折叠中间体)都结构在蛋白质的相同特定区域。RNaseH以一种明显的分层方式折叠，其中最稳定的单个元件首先折叠。这些结果表明蛋白质稳定性、结构和折叠之间的关系具有重要的意义。该提案中概述的实验探索了这项工作的含义，以了解什么使蛋白质的一个区域最稳定，以及蛋白质的整体能量格局如何影响其全球稳定性、折叠和动态。具体来说，这个建议的目的是：1.利用定点突变探索大肠杆菌RNaseH的能量格局，测试天然状态下的稳定性等级与蛋白质折叠途径(S)之间的关系。2.确定组成RNaseH折叠核心的自主折叠片段的结构、稳定性和折叠。3.确定一株嗜热性RNaseH(T.thermophilus)的能量图谱。这种蛋白质的结构、稳定性和折叠的特征将与中温同系物进行比较，看看热稳定性的一般规则是否在于蛋白质内的能量分配。

The goal of this proposal is to understand how the amino acid sequence of a protein dictates its fold and stability. In order to accomplish this goal, we need to understand the structure and stability of all the conformations accessible to a given sequence, i.e. its energy landscape. The difficult with this is that proteins are extremely cooperative. In order to bypass this problem, we have dissected a protein's structure and stability into smaller pieces and studied both the partially-folded conformations and folded parts of the protein E. coli ribonuclease H. by comparing the results from kinetic and equilibrium experiments, we have obtained evidence for a surprising conclusion: all of the partially folded conformations (the acid molten globule, the higher energy conformations of the native state and a kinetic folding intermediate) are structured in the same specific regions of the protein. RNase H folds in an apparently hierarchical fashion in which the most stable individual element folds first. These results suggest important implications about the relationship between protein stability, structure and folding. The experiments outlined in this proposal explore the implications of this work to learn what makes one region of the protein the most stable and how the overall energy landscape of a protein affects its global stability, folding and dynamics. Specifically, the aims of this proposal are: 1. Explore the energy landscape of E. coli RNase H using site-specific mutagenesis, testing the relationship between the hierarchy of stability in the native state and the folding pathways(s) of the protein. 2. Determine the structure, stability and folding of autonomous folding fragments comprising the folding core of RNase H. 3. Determine the energy landscape of a thermophilic RNase H (T. thermophilus). The feature of the structure, stability and folding of this protein will be compare to the mesophilic homologue to see if the general rules for thermostability lie in the distribution of energy within the protein.