NMR STUDIES OF THREE-DIMENSIONAL STRUCTURE OF MACROMOLECULES IN SOLUTION

该实验室的工作重点是测定 溶液中大分子的三维结构。方法 正在开发，以提高结构的精度， 确定，可以分析的蛋白质的分子量范围， 以及用于确定 结构的基础上的NMR数据。我们特别 证明了异相3D NMR对研究 蛋白质在15-25 kDa的范围内，最近，我们已经证明， 异源4D NMR在分配核Overhauser效应中的应用 这实际上是一种自动化的方式， 甚至更大的蛋白质。 许多蛋白质的高分辨率溶液结构已经被 测定这些包括细胞因子白细胞介素-8、人硫氧还蛋白和 人增强子结合蛋白的锌指结构域。广泛使用 在这些研究中，已经进行了系统的构象搜索， 获得立体定向分配和扭转角限制， 使我们能够获得更高的精度和准确性的结构， 是异源的可达到的典型精度为0.3-0.4 A， 主链原子和内部侧链为0.4-0.6 A。 目前正在进行确定若干问题的解决方案结构的工作， 其他蛋白质。这些包括来自噬菌体Mu的DNA结合蛋白ner， 人白细胞介素-lo和来自蛔虫的胰蛋白酶抑制剂。的情况下 人白细胞介素-1-β，3D和4D杂波NMR的广泛使用， 解决与光谱重叠和质子相关的问题 化学位移简并

Work in this laboratory has been focused on the determination of three-dimensional structures of macromolecules in solution by NMR. Methods are being developed to increase the precision with which structures can be determined, the molecular weight range of proteins that can be analysed, and the efficiency of the computational methods used to determine the structures on the basis of the NMR data. In particular, we have demonstrated the applicability of heteronuclear 3D NMR to the study of proteins in the range 15-25 kDa, and recently, we have demonstrated the utility of heteronuclear 4D NMR to assign nuclear Overhauser effects in virtually an automated manner which will permit the extension of the method to even larger proteins. High resolution solution structures of a number proteins have been determined. These include the cytokine interleukin-8, human thioredoxin and the zinc finger domain of a human enhancer binding protein. Extensive use in these studies has been made of systematic conformational searches to obtain stereospecific assignments and torsion angle restraints which have enabled us to obtain structure of much greater precision and accuracy than was heteretofore possible. The typical accuracy attainable is 0.3-0.4 A for the backbone atoms and 0.4-0.6 A for the internal side chains. Work is in progress on determining the solution structures of a number of other proteins. These include the DNA binding protein ner from phage Mu, human interleukin-lo and a trypsin inhibitor from Ascaris. In the case of human interleukin-1-beta, extensive use of 3D and 4D heteronuclear NMR has been made to resolve problems associated with spectral overlap and proton chemical shift degeneracy.