FOLDING OF THE ACID STATE, SUBDOMAINS AND INTACT RNASE H

这个建议的目的是绕过蛋白质的协同性 通过剖析蛋白质的结构和折叠， 加工成更小的碎片。 这将通过隔离和 表征分离折叠的蛋白质片段， 鉴定和表征所述蛋白质的部分折叠状态。 这些子域和平衡中间体将允许评估 同一条多肽链在不同的环境中。 所提出的实验中使用的模型系统是核糖核酸酶H （RNase H）酶家族。 最近的晶体结构的E。 coli RNase H和来自HIV逆转录酶的RNase H结构域揭示了 子域名的自然例子。 此外，初步研究表明， 又发现了两个子域一个15个残基长螺旋亚结构域， 另一个通过有限的蛋白水解分离， 蛋白 蛋白质的平衡中间状态类似于 “熔球”也被揭开了面纱。 拟议的实验 描述这些替代物的热力学和结构分析 多肽链的形式及其相互关系。 具体而言，该提案的目的是： 1. 确定肽的结构、稳定性和折叠 通过RNase H的有限蛋白水解获得的片段或亚结构域 链霉蛋白酶 2. 为了表征螺旋形成的结构和稳定性， 衍生自蛋白质的螺旋E的肽。 基因突变的影响 分离的肽和完整蛋白上的螺旋将 评估。 3. 为了表征核糖核酸酶H的部分折叠的酸状态， 并测试是否存在类似于部分折叠酸状态的状态 作为核糖核酸酶折叠途径中的一种短暂的中间体 H.

The goal of this proposal is to bypass the cooperativity of protein folding and stability by dissecting a protein's structure and folding process into smaller pieces. This will be done by isolating and characterizing fragments of the protein that fold in isolation,a nd by identifying and characterizing partially-folded states of the protein. These subdomains and equilibrium intermediates will allow an evaluation of the same polypeptide chain in different contexts. The model system used in the proposed experiments is the ribonuclease H (RNase H) family of enzymes. The recent crystal structures for both E. coli RNase H and the RNase H domain from HIV reverse transcriptase reveal natural examples of subdomains. In addition, preliminary studies have revealed two more subdomains. one 15 residue long helical subdomain and another isolated by limited proteolysis containing approximately half the protein. An equilibrium intermediate state of the protein similar to the "molten globule" has also been uncovered. The proposed experiments describe a thermodynamic and structural analysis of these alternative forms of the polypeptide chain and their relationship to each other. Specifically, the aims of this proposal are: 1. To determine the structure, stability and folding of the peptide fragments, or subdomain(s), obtained by limited proteolysis of RNase H with pronase. 2. To characterize the structure and stability of the helix-forming peptide derived from helix E of the protein. The effect of mutations in this helix on the isolated peptide and the intact protein will be evaluated. 3. To characterize the partially-folded acid state of Ribonuclease H, and to test if a state similar to the partially-folded acid state exists as a transiently populated intermediated in the folding pathway of RNase H.