THIOREDOXIN AND GLUTAREDOXIN STABILITY AND FUNCTION

我们建议继续研究稳定性， 结构和功能的相关蛋白，E.大肠杆菌硫氧还蛋白和 谷氧还蛋白是一个重要且普遍存在的 氧化还原酶 在这两种蛋白质中，氧化还原功能由 分子中仅有的两个半胱氨酸的二硫二硫醇反应。 我们正在研究特定残基在氧化还原活性中的作用 和谷氧还蛋白的折叠。 这些残基是Cys 32 和硫氧还蛋白中的Cys 35（谷氧还蛋白中的11和14）， 硫氧还蛋白（谷氧还蛋白中的Pro 60）和硫氧还蛋白中的Asp 26（ 是脂肪族残基）。 在硫氧还蛋白中， 以下过程是链接的功能：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 这些残基是完全保守的同源 proteins. 我们有三个具体目标，为此我们使用了一些 生物物理技术，并采用硫氧还蛋白的突变体， 谷氧还蛋白。 1)阐明了热力学与整体稳定性的联系， 特定基团的电离，脯氨酸肽异构和活性 二硫化物-二硫醇氧化还原平衡。 2)检查氧化还原机制，特别是 硫氧还蛋白中活性位点半胱氨酸-SH的扰动pKa值， 谷氧还蛋白和硫氧还蛋白中的Asp26。 3)比较和对比 硫氧还蛋白相对于谷氧还蛋白的1）和2）过程，以及 谷氧还蛋白与谷氧还蛋白-N。 本研究将使用的生物物理技术包括 差示扫描量热法，拉曼光谱 和圆二色性光谱。

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.