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Application of Segmental Isotope Labeling Method for Protein NMR and Free Energy Calculation for Development of Inhibitors for Proteins.

应用分段同位素标记方法进行蛋白质核磁共振和自由能计算以开发蛋白质抑制剂。

基本信息

批准号：
12558083
负责人：
NAKAMURA Haruki
金额：
$ 8.58万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Our purpose is to build a precise structural model of a protein-ligand complex by combining the segmental isotope labeling method for protein NMR and the free energy calculation. It can be applied to development of new drugs for target proteins.As a preliminary experiment, we introduced the segmental isotope label into the maltose binding protein (MBP : 370 a.a.), and observed NMR signals. However, when the ligands were added to the protein solution, very many new resonace peaks appeared, in addition to few chemical shift changes. Thus, we concluded that MBP is not a suitable system for the current purpose. Instead, we used the b-subunit of ATP synthase, and a part of this enzyme (391-473) was labeled by ^<15>N using the segmental isotope labeling method. When the NMR signal of ^<15>N was observed, almost all the resonance peaks were identified as separated signals. By adding ADP and Mg^<2+> to this solution, we obtained the new information corresponding to the structural changes in the protein.As a consequence of investigation for 92 enzyme families for the target of drug design, D-alanyl-D-alanine peptidase (VanX : about 200 a.a.) was found to be a good candidate. We synthesized an inhibitor of VanX, and prepared the uniform labeled VaxX by ^<15>N and ^<13>C after establishing the overexpression system using E. coli. When the synthesized inhibitor was added to the VanX solution, several particular chemical shifts of the NMR peaks changed significantly, so that the active site can be identified.Simultaneously, the free energy change was estimated by docking simulation using the multicanonical WHAM upon the ligand binding to a protein. In addition, the interface between the protein and the ligand was analyzed by the saturation transfer. NMR experiment and by solving the Bloch equation during the simulated annealing. We applied this new method to the system of the CAD-ICAD complex.

我们的目的是建立一个精确的结构模型的蛋白质-配体复合物相结合的片段同位素标记方法的蛋白质NMR和自由能计算。作为初步实验，我们将片段同位素标记引入麦芽糖结合蛋白（MBP：370 a.a.），并观察到NMR信号。然而，当配体被添加到蛋白质溶液中时，除了很少的化学位移变化之外，还出现了非常多的新的共振峰。因此，我们得出结论，MBP不是一个适合当前目的的系统。相反，我们使用ATP合酶的b亚基，并使用片段同位素标记方法用^ N标记该酶的一部分（391-473）<15>。当观察^ N的NMR信号时<15>，几乎所有的共振峰都被识别为分离的信号。通过对92个酶家族的药物设计研究，筛选出D-丙氨酰-D-丙氨酸肽酶（VanX：约200 a.a.）他被认为是一个很好的候选人。我们合成了VanX的抑制剂，并<15><13>利用E.杆菌当合成的抑制剂加入到VanX溶液中时，NMR谱中几个特定的化学位移发生了明显的变化，从而可以确定活性位点，同时，通过多正则WHAM对接模拟计算了配体与蛋白质结合时的自由能变化。此外，通过饱和转移分析了蛋白质与配体之间的界面。NMR实验和模拟退火过程中求解Bloch方程。我们将这种新方法应用于CAD-ICAD系统中。