PROTEIN NMR STRUCTURE AND DYNAMICS VIA ISOTOPIC LABELING

一些小蛋白质的主链上的内部流动性 通过1H-检测的15N核磁共振弛豫实验进行了监测。扩展 这种处理含质子碳的方法将增强 对蛋白质动力学的理解不仅仅是因为更大的数字 还通过监控侧链动态，为其提供了更广泛的 预计内部机动性将达到一定范围。不幸的是，这样的13C 松弛实验因两个主要障碍而变得复杂。虽然 除了最小的蛋白质系统外，同位素标记在所有系统中都是必要的， 均匀浓缩产生同核标量和偶极 相互作用扰乱了标准的松弛实验。 此外，偶极-偶极干扰效应严重地使 亚甲基和甲基共振弛豫数据的解释 尽管对于对称甲基的情况存在部分解 亚甲基案件仍然是一个有问题的系统。 这两个障碍都可以通过将两个同位素相结合来克服 本实验室以前开发的标签图案，交替 碳的富集度和随机分数的重氢。在第一个标签中 每种残基类型都富含13C-12C-13C…花样输出 从而在没有一个的情况下提供了高水平的浓缩 粘合13C-13C联轴器。随机分数重氢与 多重编辑序列和2H门控去耦合可以产生抑制 来自含两个质子的碳的信号，因此单质子信号 可以获得和分析类似于亚胺和主链酰胺的化合物 共鸣。将对大肠杆菌硫氧还蛋白进行松弛分析 和葡萄球菌核酸酶。这两种蛋白质都有很高的分辨率 X-射线结构以及核磁共振归属和结构研究。E. Coli硫氧还蛋白已被用来显示这种标记的实用性 方法和初步研究定性地指出了动力学的范围 现在时。将进行定量的动力学分析。越大 葡萄球菌核酸酶是生物物理和生物化学的首选模型系统 蛋白质折叠研究的遗传学方法。这场运动的动向 核磁共振显示Apo与PTP抑制酶应提供洞察力 底物类似物结合诱导的强直化是如何传播的 通过蛋白质结构。 随机分数氚有效地抑制了自旋扩散 NOE距离估计的影响。这些NOE的定量比较 与高分辨率x射线结构的距离将提供有用的 核磁共振约束条件可靠性评估的统计学基础 确定蛋白质结构的局部和全局方面。使用 约束的统计权重将显著澄清当前 在定义和评估结构中的目标函数时的含糊性 精炼过程。预计将提供更多 精确的NOE距离约束以及相关的统计基础 将有助于解决因比较而产生的残余歧义 溶液和晶体结构，并增强了对 缺少相应X射线分析的体系的溶液结构。

Internal mobility along the mainchain of a number of small proteins has been monitored using 1H-detected 15N NMR relaxation experiments. Extending this approach to the proton bearing carbons would enhance the understanding of protein dynamics not only due to the much larger number of sites, but also by monitoring sidechain dynamics for which a wider range of internal mobility is anticipated. Unfortunately, such 13C relaxation experiments are complicated by two major impediments. Although isotope labeling is necessitated in all but the smallest protein systems, uniform enrichment gives rise to homonuclear scalar and dipolar interactions which confound the standard relaxation experiments. Furthermore, dipole-dipole interference effects severely complicate the interpretation of relaxation data of methylene and methyl resonances. Although partial solutions exist for the case of the symmetric methyl system, the methylene case remains problematic. Both of these impediments can be overcome by combining two isotopic labeling patterns previously developed in this laboratory, alternating carbon enrichment and random fractional deuteration. In the first labeling pattern 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. Random fractional deuteration combined with multiplet editing sequences and 2H gated decoupling can yield suppression of signals from carbons bearing two protons so that the monoprotio signal can be obtained and analyzed analogous to the methine and mainchain amide resonances. Relaxation analysis will be carried out on E. coli thioredoxin and staphylococcal nuclease. Both proteins have reported high resolution x-ray structures as well as NMR assignments and structural studies. E. coli thioredoxin has been used to show the practicality of this labeling approach and initial studies qualitatively indicated the range of dynamics present. Quantitative dynamical analyses will be carried out. The larger staphylococal nuclease is the premiere model system for biophysical and genetic approaches to the study of protein folding. The dynamics of the apo vs. pTp inhibited enzyme as revealed by NMR should provide insight into how the rigidification induced by substrate analog binding propagates through the protein structure. Random fractional deuteration effectively suppresses spin diffusion effects in NOE distance estimates. Quantitative comparison of these NOE distances with the high resolution x-ray structure will provide a useful statistical basis for assessing the reliability of NMR constraints in determining both local and global aspects of protein structure. Use of statistical weighting of constraints will markedly clarify present ambiguities in defining and assessing target functions in the structure refinement process. It is anticipated that the availability of more accurate NOE distance constraints along with the related statistical base will help resolve residual ambiguities resulting from comparisons between solution and crystal structures as well as enhance the confidence in solution structures of systems lacking corresponding x-ray analyses.