NMR OF STAPHYLOCOCCAL NUCLEASE & DYNAMICS IN SOLUTION STRUCT DETERMINATION

金黄色葡萄球菌核酸酶的 NMR

• 批准号: 6309117
• 负责人: DAVID M LEMASTER
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309117
• 关键词: biological products; biomaterials; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

We have developed a biosynthetic method for generating protein samples in which all of the amino acid residues have an alternating 13C-12C-13C. pattern for nearly all positions. This pattern serves to eliminate the scalar and dipolar interactions between adjacent 13C nuclei which severely complicates the extraction of dynamical data from relaxation measurements. When this pattern is combined with random fractional deuteration the signals from the carbons bearing a single proton can be selected, thus eliminating the interference between the 1H-13C 1H-13C dipolar interference effects which complicates relaxation analysis of methylene and methyl positions. We have used this combined labeling approach to analyze the dynamics of nearly all proton bearing carbons of E. coli thioredoxin. Far more detailed analysis of sidechain dynamics has been obtained than that previously reported. The combination of mainchain and sidechain relaxation data has led to structural interpretation of the millisecond conformational transitions observed in several regions of the molecule. In particular, structural interpretation of the dynamics at the active site has led to a model for dynamical coupling to the catalytic transition. Independently NOE buildup measurements have been carried out on E. coli thioredoxin random fractionally deuterated to 75%. We have demonstrated the effective suppression of spin diffusion by this labeling pattern. As spin diffusion and internal mobility are commonly regarded to be the primary causes of inaccuracies in NOE distance constraints, the combined relaxation and NOE measurements have been used to interpret the detailed differences between the independently determined solution and x-ray structures of E. coli thioredoxin. We propose to carry out analogous relaxation and NOE buildup experiments on the inhibited and unligated forms of staphylococcal nuclease in order to gain insight into the internal dynamics of this enzyme and their relevance to the catalytic function.

我们已经开发出一种生物合成方法来产生蛋白质