THIOREDOXIN AND GLUTAREDOXIN STABILITY AND FUNCTION

我们建议继续我们对稳定性关系的研究， 大肠杆菌硫氧还蛋白和相关蛋白的结构与功能 一个重要且普遍存在的谷氧还蛋白家族的原型 氧化还原酶。在这两种蛋白质中，氧化还原功能都是通过 二硫代二硫醇反应分子中仅有两个半胱氨酸。 我们正在研究特定残基在氧化还原活性中的作用。 硫氧还蛋白和谷氧还蛋白的折叠。这些残基是Cys 32 硫氧还蛋白为Cys 35(谷氧还蛋白为11和14)，Pro为76 硫氧还蛋白(谷氧还蛋白中的Pro 60)和硫氧还蛋白中的天冬氨酸26(其 谷氧还蛋白是一种脂肪族残留物)。在硫氧还蛋白中，我们已经证明 以下进程是链接函数：1)Cys 32和Cys 35 氧化态，2)Asp 26电离态，3)Pro 76异构体，和4) 全球稳定。这些残基在同源的 蛋白质。我们有三个具体目标，为此我们使用了一些 生物物理技术，并使用硫氧还蛋白和硫氧还蛋白的突变体 谷氧还蛋白。 1)阐明了全球稳定性的热力学联系， 特定基团的电离、脯氨酸多肽的异构性和活性 位置二硫化物-二硫醇氧化还原平衡。 2)研究氧化还原机制，特别是 硫氧还蛋白和硫氧还蛋白中活性部位半胱氨酸-SH的微扰pKa值 谷氧还蛋白和硫氧还蛋白中的天冬氨酸26。 3)比较和对比结构/功能关系 硫氧还蛋白与谷氧还蛋白的1)和2)过程，以及 谷氧还蛋白与谷氧还蛋白-N的比较 将用于这项研究的生物物理技术包括 异核核磁共振、差示扫描量热、拉曼光谱 和圆二色光谱。

We propose to continue our study of the relationships of stability, structure and function in the related proteins, E. coli thioredoxin and glutaredoxin prototypes of an important and ubiquitous family of oxidoreductases. In both proteins the redox function is mediated by disulfide dithiol reaction of the only two cysteines in the molecule. We are investigating the role of specific residues in the redox activity and folding of thioredoxin and glutaredoxin. These residues are Cys 32 and Cys 35 in thioredoxin (11 and 14 in glutaredoxin), Pro 76 in thioredoxin (Pro 60 in glutaredoxin), and Asp 26 in thioredoxin (which in glutaredoxins is an aliphatic residue). In thioredoxin, we have shown that the following processes are linked functions: 1) Cys 32 and Cys 35 oxidation state, 2) Asp 26 ionization state, 3) Pro 76 isomerism, and 4) global stability. These residues are completely conserved in homologous proteins. We have three specific aims, toward which we use a number of biophysical techniques, and employ mutants of thioredoxin and glutaredoxin. 1) Elucidation of the thermodynamic linkage of global stability, ionization of specific groups, proline peptide isomerism and the active site disulfide-dithiol redox equilibrium. 2) Examination of the redox mechanisms, particularly the roles of perturbed pKa values of the active site cysteine-SH in thioredoxin and glutaredoxin, and Asp 26 in thioredoxin. 3) Compare and contrast the structure/function relationships in the processes of 1) and 2) in thioredoxin versus glutaredoxin, and glutaredoxin versus glutaredoxin-N. The biophysical techniques to be used for this study include heteronuclear NMR, differential scanning calorimetry, Raman spectroscopy and circular dichroism spectroscopy.