PROTEIN NMR STRUCTURE AND DYNAMICS VIA ISOTOPIC LABELING

描述(改编自摘要)：首次有效监测 据报道，蛋白质中几乎所有含质子的碳都是动力学的。 通过核磁共振弛豫测量。这种生物合成标记协议产生了 每种残基类型都富含13C-12C-13C图案 因此，侧链在没有单键13C-13C的情况下提供了高水平的浓缩 联轴器，因此非常适合松弛研究。一种动力学分析 形式主义已经发展起来，它有力地解释了蛋白质的内部运动 根据任意多指数自相关函数。这个 表征局部相关运动的合成能力及其 构象熵将被应用于N-末端结构域 钙依赖信号蛋白钙调蛋白与较大的23 kDa大肠杆菌 腺苷酸激酶。钙调蛋白侧链动力学的定量分析 将在之前的主链松弛研究的基础上进行大幅扩展，以便 更清楚地了解钙结合的协同性是如何 与构象转变的动力学有关，这产生了 活动信令状态。两种底物中的每一种与ATP和AMP结合 腺苷酸激酶导致活性部位周围的大规模裂隙闭合。 这两个裂隙闭合转变之间的动态串扰程度 是一个值得积极辩论的问题。活性部位内的残基似乎 根据底物结合和强大的动力学显著改变它们的迁移率 已经假设了与域闭合的耦合。建议的放宽措施 实验将定量地解决这些关键问题。放松 测量将由剩余偶极耦合实验补充，以 提供全球定向信息，以确定各个铰链的特征 波动。弛豫和偶极耦合实验将 通过将手性氢与替代碳结合而促进的 在过氚本底中甘油碳源的浓缩。这 标记法产生了一大组孤立的1H-13C自旋对 表现出极佳的分辨率和灵敏度，适用于 弛豫和偶极耦合实验。大量的立体选择 赋值直接从丰富模式中提供。这个标签 模式同样非常适合更传统的基于NOE的解决方案 将演示结构的确定及其使用范围

DESCRIPTION (Adapted from abstract): For the first time efficient monitoring of dynamics is reported for essentially all proton-bearing carbons in a protein via NMR relaxation measurements. This biosynthetic labeling protocol generates each residue type is enriched with a 13C-12C-13C pattern out along each sidechain thus providing high levels of enrichment without one bond 13C-13C couplings and thus well suited for relaxation studies. A dynamics analysis formalism has been developed that robustly interprets protein internal motion in terms of an arbitrary multiexponential autocorrelation function. The resultant ability to characterize local correlated motion and its conformational entropy will be applied to the N-terminal domain of the calcium-dependent signal protein calmodulin and to the larger 23 kDa E. coli adenylate kinase. Quantitative analysis of the sidechain dynamics of calmodulin will substantially expand upon previous mainchain relaxation studies so as to provide a clearer understanding of how the cooperativity of calcium binding is related to the dynamics of the conformational transition, which yields the active signaling state. Binding of each of the two substrates ATP and AMP to adenylate kinase results in large scale cleft closures around the active site. The degree of dynamical crosstalk between these two cleft closure transitions is matter of active debate. Residues within the active site appear to substantially change their mobility upon substrate binding and strong dynamical coupling to the domain closure has been postulated. The proposed relaxation experiments will quantitatively address these key issues. The relaxation measurements will be complemented by residual dipolar coupling experiments to provide global orientational information to characterize the individual hinge fluctuations. The relaxation and dipolar coupling experiments will be facilitated by combining chiral deuteration with the alternate carbon enrichment of the glycerol carbon source in a perdeuterated background. This labeling approach gives rise to a large set of isolated 1H-13C spin pairs exhibiting excellent resolution and sensitivity suitable for both the relaxation and dipolar coupling experiments. A large number of stereoselective assignments are provided directly from the enrichment pattern. This labeling pattern is likewise well suited for more conventional NOE-based solution structure determinations and its range of utility will be demonstrated