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性、