核磁共振研究蛋白质内含子的溶液性质与蛋白剪接机理

项目名称： 核磁共振研究蛋白质内含子的溶液性质与蛋白剪接机理

项目编号： No.20873135

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 刘扬中

作者单位： 中国科学技术大学

项目金额： 36万元

中文摘要： '蛋白质内含子'（Intein）在蛋白工程中被广泛应用，但其机理研究较少；另一方面，Intein为结核菌生长所必须，因而被认为新型是药物设计靶点。本项目以二维、三维核磁共振(NMR)为主要手段，研究Intein的溶液性质与蛋白剪接机理。运用分子生物学手段，制备了13C 15N 同位素标记的Intein蛋白质以及多种突变体，通过NMR解析蛋白质结构，运用NMR技术结合蛋白质突变研究特定氨基酸残基的溶液性质，通过pH 滴定检测蛋白质活性中心的组氨酸和天冬氨酸侧链的pKa、氨基酸的氢键状态，通过R1、R2、NOE等实验研究关键氨基酸突变对蛋白质动力学性质的影响，从而阐明Intein的剪接机理。 在Intein结构与机理研究的基础上，我们采用NMR技术、结合ITC等手段研究金属离子与Intein 的作用，掌握金属性质、配位方式以及热力学参数对金属离子对Intein 抑制作用的关系，得到了在体外、体内均具有Intein活性抑制作用的铂配合物，这一配合物通过抑制Intein剪接抑制结核菌的生长。同时，进一步从反应动力学、配位结构等方面研究了铂配合物与Intein蛋白质的相互作用，以阐明抑制剂机理。

中文关键词： 核磁共振；溶液结构；动力学性质；蛋白质剪接；抑制剂

英文摘要： Although Intein has widely used in protein engineering, their mechanism study is still limited. Other the other hand, Intein is required for TB proliferation and is considered as a novel drug target. In this project, we performed various 2D and 3D NMR to study the solution property of Intein and the splicing mechanism. We have solved the protein structure, studied the dynamics property (R1, R2, NOE, CPMG) of several mutants, measured the pKa of histidine and aspartate in catalytic core. We found the H-bonds induced by special pKa of key residues play crucial roles in the protein splicing. Based on the mechanism studies, we further investigated the interaction of intein with metal ions. By using NMR, ITC, EXAFS, we rationalized the coordination property to the inhibition efficacy. We found that metal ions inhibit protein splicing by binding to key residues in intein and restraining their dynamic property, which is required for peptide bond rearrangements. Among a number of metal complexes, cisplatin demonstrated the best inhibitory activity both in vitro and in vivo. Experiments on TB cells showed that cisplatin can inhibit the growth of TB cells. This finding provides a novel approach for TB-drug development. We also performed NMR, MS and fluorescence measurement to study the mechanism of the reaction between intein and cisplatin. Results indicate that cisplatin can bind to Cys1, directly prevents the nucleophilic attack of thiol group.

英文关键词： NMR; structure; dynamics; protein splicing; inhibitors