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

我们的目的是将蛋白质核磁共振的分段同位素标记法与自由能计算相结合，建立蛋白质-配体复合体的精确结构模型。作为初步实验，我们将节段性同位素标记引入到麦芽糖结合蛋白(MBP：370 A.A.)中，并观察到核磁共振信号。然而，当配体加入到蛋白质溶液中时，除了很少的化学位移变化外，还出现了许多新的共振峰。因此，我们得出结论，MBP不是一种适合当前目的的制度。相反，我们使用了ATP合成酶的b亚基，并使用分段同位素标记方法将该酶的一部分(391-473)用^&lt；15&gt；N标记。当观察到^&lt；15&gt；N的核磁共振信号时，几乎所有的共振峰都被鉴定为分离的信号。通过加入ADP和镁离子，我们得到了与蛋白质结构变化相对应的新信息。作为药物设计靶点的92个酶家族的研究结果，D-丙氨酰-D-丙氨酸肽酶(VanX：约200A.A.)被认为是一个很好的候选人。我们合成了一种VanX的抑制剂，并利用大肠杆菌建立了过量表达系统，制备了由^&lt；15&gt；N和^&lt；13&gt；C统一标记的Vaxx。当合成的抑制剂加入到VanX溶液中时，核磁共振谱峰的几个特殊的化学位移发生了显著的变化，从而可以识别活性中心。同时，通过对接配体与蛋白质结合的多峰WHAM模拟估计了自由能的变化。此外，通过饱和转移分析了蛋白质与配体之间的界面。核磁共振实验，并在模拟退火法中求解Bloch方程。我们将这种新方法应用于CAD-ICAD复合体系统。

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.