FOLDING OF THE ACID STATE, SUBDOMAINS AND INTACT RNASE H

该提案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访问的所有构象的结构和稳定性，即其能量景观。困难的是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分解为较小的片段，并研究了蛋白质E的部分折叠构象和折叠部分。大肠杆菌核糖核酸酶H通过比较动力学和平衡实验的结果，我们得到了一个令人惊讶的结论的证据：所有的部分折叠构象（酸熔融球、天然状态的较高能量构象和动力学折叠中间体）都在蛋白质的相同特定区域中结构化。RNase H以明显的分级方式折叠，其中最稳定的单个元件首先折叠。这些结果表明蛋白质的稳定性，结构和折叠之间的关系的重要影响。该提案中概述的实验探索了这项工作的意义，以了解是什么使蛋白质的一个区域最稳定，以及蛋白质的整体能量景观如何影响其全局稳定性，折叠和动力学。具体而言，本提案的目的是：1.探索E的能源景观。coli RNase H，测试天然状态下的稳定性等级与蛋白质的折叠途径之间的关系。2.确定包含RNase H折叠核心的自主折叠片段的结构、稳定性和折叠。3.确定嗜热RNA酶H（T。嗜热菌）。该蛋白质的结构、稳定性和折叠的特征将与嗜温同系物进行比较，以了解热稳定性的一般规则是否在于蛋白质内的能量分布。

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.