NMR OF STAPHYLOCOCCAL NUCLEASE & DYNAMICS IN SOLUTION STRUCT DETERMINATION

我们已经开发出一种生物合成的方法， 其中所有氨基酸残基具有交替的氨基酸残基的样品 13C-12C-13C 几乎所有职位的模式。 这种模式服务于 以消除相邻13 C之间的标量和偶极相互作用 原子核，这使得动力学数据的提取变得非常复杂 从弛豫测量。 当这种模式与 随机分数氘化来自带有a的碳的信号 可以选择单个质子，从而消除干扰 1H-13 C 1H-13 C偶极干涉效应之间， 使亚甲基和甲基位置的弛豫分析复杂化。 我们 使用这种组合标记方法来分析 几乎所有的质子携带的碳E。大肠杆菌硫氧还蛋白。 得多 详细分析了侧链动力学， 此前报道。 主链和侧链的结合 松弛数据导致了结构解释的 毫秒构象转换观察到的几个地区， 分子。 特别是，结构的解释， 在活性位点的动力学导致了动力学耦合的模型 到催化转变。 独立NOE积聚测量 在E.大肠杆菌硫氧还蛋白随机分数 氘含量达到75% 我们已经证明了有效抑制 通过这种标记模式进行自旋扩散。 由于自旋扩散和 内部流动通常被认为是主要原因， NOE距离约束的不准确性， NOE测量已被用于解释详细的差异 在独立确定的溶液和X射线结构之间， E.大肠杆菌硫氧还蛋白。 我们建议进行类似的放松， NOE的建立实验中， 葡萄球菌核酸酶，以便深入了解内部 这种酶的动力学及其与催化功能的相关性。

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.