本团队将针对指南方向1：生物大分子动态修饰的化学标记与检测技术开展研究。G-蛋白偶联受体(GPCR)是30%以上的药物靶点，其信号转导受到磷酸化的精确调控。GPCR下游传导蛋白虽然只有十几种G蛋白和4种arrestin蛋白，却能够传递极为丰富的生理信号的重要机制是GPCR的C端尾巴的磷酸化编码。通过开发基因密码子扩展方法，我们拟进行以下研究：1.配体结合GPCR后，产生了何种动态构象变化使得GPCR激酶(GRK)被激活，使得GPCR被磷酸化？2.在arrestin，GRK特定位点引入探针，研究激活的GPCR诱导arrestin，GRK产生动态构型变化的机理。3.通过动态共价键的引入，制备GPCR/GRK的稳定复合物，解析结构。以上研究体系的建立，有望为激酶和底物蛋白相互作用机理研究开辟一条新途径，并解析GPCR偏向性信号转导机制，为研发高效低副作用药物打下基础。

In this proposal, we will aim for the study of biomacromolecule labelling and detection methods. G-protein coupled receptor (GPCR) is the drug target of more than 30% pharmaceuticals. The downstream signal of GPCR is precisely regulated by phosphorylation. While there are only about a dozen G protein and 4 arrestin proteins which serve as transducer proteins of GPCR, activation of GPCR can result in more than 10,000 distinct physiological signals. An underlying mechanism for this diverse signaling is the phospho-barcode present in the C-terminus of GPCR. Through the development of genetic code expansion method, we will perform the following studies. First, we will study the dynamic conformation change of GPCR (in particular the cannabinoid receptor) in response to the binding of agonist, and how dynamic conformation change in GPCR lead to GPCR kinase (GRK) activation. Second, we will introduce NMR and fluorescence probes site-specifically into arrestin and GRK, to investigate how activated GPCR triggers conformation changes in arrestin and GRK, leading to their activation. Third, through the introduction of dynamic covalent bond, we will prepare stable complex of GPCR and GRK, and solve the complex structure using cryo-EM and other methods. The establishment of the above methods will establish a new route for the systematic investigation of kinase/substrate protein interaction mechanism, delineate biased signaling mechanism of GPCR signal transduction, and lay a solid foundation for the design of drugs which have high efficacy and low side effects.