基于基因密码子扩展的磷酸化蛋白质合成

蛋白质磷酸化是最重要的蛋白质翻译后修饰之一。磷酸化作为细胞内信号转导的关键机制，能够精确的调控蛋白质的构象、相互作用、酶活。蛋白质的动态磷酸化-去磷酸化循环是细胞信号传递的主要途径。蛋白质磷酸化研究的主要瓶颈是如何大量获得具有特定位点磷酸化修饰的蛋白。本研究旨在将化学生物学的方法应用到蛋白质磷酸化研究中，通过发展基因编码扩展技术，设计系列磷酸化氨基酸，实现其高效有机合成；通过正负筛选的方法获得对其特异性识别的氨酰-tRNA合成酶；将磷酸化氨基酸特异性插入目标蛋白中具有磷酸化修饰的位点。在大量获得特定位点磷酸化蛋白的基础上，我们将用电镜、晶体学等方法研究磷酸化对蛋白质结构的调控，利用核磁共振等方法研究蛋白质构象、活性及与其它大分子动态相互作用。该研究将为探索体内磷酸化氨基酸修饰的调控方式及信号传递过程提供一种研究技术手段，为设计新的抗体及药物提供研究基础。

Protein phosphorylation is one the most important post-translational modifications. As a key mechanism for cellular signal transduction, protein phosphorylation can precisely regulate protein conformation, interaction and enzyme activity. The dynamic phosphorylation/dephosphorylation is the major mechanism for cellular signal transduction. The main bottleneck for protein phosphorylation study is how to obtain large quantity (more than milligram) of proteins bearing site-specific phosphorylation. In this study, we aim to apply chemical biology methods for the study of protein phosphorylation. Specifically, we will further develop the genetic code expansion methods, synthesize a library of phosphor-amino acids, obtain the tRNA synthetases which specific recognize these unnatural amino acids, and then efficiently incorporate phosphor-amino acids into target protein. Once we obtain large quantity of proteins bearing site-specific phosphorylation, we will use structural biology methods such as X-ray crystallography, cryo-EM to investigate how phosphorylation regulates protein structure, and use biophysical methods such as NMR to investigate the dynamic conformation change of proteins. This study will provide a powerful new method for investigating protein phosphorylation, and provide basis for preparing antibodies and drug discovery.