THIOREDOXIN AND GLUTAREDOXIN STABILITY AND FUNCTION

硫氧还蛋白和谷氧还蛋白的稳定性和功能

• 批准号: 2185682
• 负责人: CLARE K WOODWARD
• 金额: $ 17.63万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185682
• 关键词: Raman spectrometry; acidity /alkalinity; active sites; biophysics; calorimetry; chemical stability; chemical synthesis; circular dichroism; cis trans isomerization; cofactor; disulfide bond; dithiol; enzyme mechanism; hydrogen transport; ionization constant; isomer; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; oxidoreductase; protein folding; protein structure function; redoxin; thermodynamics; thioredoxin

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.

我们建议继续研究稳定关系， 相关蛋白质的结构和功能，大肠杆菌硫氧还蛋白和 重要且无处不在的家族的谷蛋白原型 氧化还原酶。 在这两种蛋白质中，氧化还原功能均由 分子中仅有两个半胱氨酸的二硫这二硫醇反应。 我们正在研究特定残基在氧化还原活性中的作用 以及硫氧还蛋白和谷蛋白的折叠。 这些残留物是Cys 32 和硫氧还蛋白（谷糖11和14）中的Cys 35，Pro 76 硫氧还蛋白（谷氨酸蛋白中的Pro 60）和硫氧还蛋白的ASP 26（它们 在谷胱甘肽中，是脂肪族残基）。 在硫氧还蛋白中，我们已经显示 以下过程是链接的函数：1）CYS 32和CYS 35 氧化态，2）ASP 26电离状态，3）Pro 76异构主义和4） 全球稳定性。 这些残基在同源物中完全保守 蛋白质。 我们有三个特定的目标，我们使用多个目标 生物物理技术，并采用硫氧还蛋白的突变体和 谷毒素。 1）阐明全球稳定性的热力学连接， 特定组的电离，脯氨酸肽异构主义和活性 位点二硫这二基氧化还原平衡。 2）检查氧化还原机制，尤其是 硫氧还蛋白和活性位点半胱氨酸SH的扰动PKA值和 甲状腺素中的谷物毒素和硫氧还蛋白的ASP 26。 3）比较和对比结构/功能关系 1）和2）在硫氧还蛋白与谷氨酸毒素中的过程，以及 谷毒素与谷糖蛋白-N。 本研究的生物物理技术包括 异核NMR，差分扫描量热法，拉曼光谱法 和圆形二色性光谱。