NMR OF STAPHYLOCOCCAL NUCLEASE & DYNAMICS IN SOLUTION STRUCT DETERMINATION

我们开发了一种产生蛋白质的生物合成方法。 所有氨基酸残基都有交替排列的样品 13C-12C-13C。几乎所有头寸的模式。这种模式服务于 消除相邻13C之间的标量和偶极相互作用 使动力学数据的提取严重复杂化的原子核 来自松弛测量。当此模式与 随机分数氢，来自碳的信号带有一个 可以选择单个质子，从而消除了干扰 在1H-13C、1H-13C偶极干扰效应之间 使亚甲基和甲基位置的松弛分析复杂化。我们 已经使用这种组合标记方法来分析 几乎所有的含质子碳的大肠杆菌硫氧还蛋白。多得多 对侧向链条的动力学进行了详细的分析。 之前报道过。主链和侧链的结合 松弛数据导致市场对 在几个区域中观察到的毫秒构象转变 分子。特别是，对 活性中心的动力学导致了动力学耦合的模型 到催化转化期。独立的NOE积聚测量 对大肠杆菌硫氧还蛋白进行了随机分次检测 氚含量降至75%。我们已经证明了有效地抑制了 通过这种标记模式的自旋扩散。因为自旋扩散和 内部流动通常被认为是导致 NOE距离约束的不精确度、组合松弛和 NOE测量被用来解释详细的差异 在独立确定的溶液和x射线结构之间 大肠杆菌硫氧还蛋白。我们建议进行类似的放宽和 抑制型和非结合型的NOE聚集实验 为了洞察葡萄球菌核酸酶的内部 该酶的动力学及其与催化功能的关系。

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.