Fluorescence Lifetime And Cryo-EM To Investigate The Activation Of ?₂AR On Downstream G Protein

G protein-coupled receptors(GPCRs)are the most diverse and functional super-family receptors in the mammalian genome.GPCRs recognize a varity of extracellular stimuli and regulate many physiological activities in organisms by activating specific downstream signaling pathways.According to statistics,GPCRs have developed into the largest drug target in the human body.There are thousands of types of GPCRs in organisms,but the downstream signaling pathways mediated by GPCRs are very conserved.All GPCRs are functional through four types of G proteins,included Gs,Gi/o,Gq/11,and G12/13.G protein signal ingestion system is a very complex network system,and the study of G protein activation is also a hot and difficult problem in the field of GPCRs.At present,nearly a hundred kinds of GPCRs and more than a dozen GPCR-G protein complex crystal structure or electron microscopic structure has resolved,but dynamic conformation changes of G-protein relatively less.Therefore,it is significant to study how the signal passed and how dynamic conformation changes of G protein after it was activated.Time-resolution fluorescence technology can reflect the possible conformations and their distribution of proteins in the complex systems,and compared with the detection of fluorescence intensity in the steady-state spectrum,time-resolution fluorescence lifetime detection is not affected by light intensity,light path scattering,probe concentration,etc.Therefore,it is a good method for studying protein conformation and conformational changes.The first part of this paper,we applicated time-resolution fluorescence technology,combining with the good fluorescence characteristics and the advantages of site-specific labeling of fluorescent non-natural amino acid 7-HC to explore the conformation changes of the G alphai1 protein in the activation process.By measuring the fluorescence lifetime and anisotropy,combinating with the crystal structure analysis to expound the molecular basis of G protein activation.The second part experiment,we also applied fluorescence lifetime analysis and fluorescence lifetime FRET method to analyze the different conformational distributions of G?s and G?i1 proteins and to explore the conformational changes between this two G proteins in response to ?2AR activation.Breaking the current situation of the structure study of ?2AR-G protein complex is limited to ?2AR-Gs protein complex.It is proposed that?2AR has different activation effects on Gas and G?i1 proteins.In the third part of the experiment,we combined Cryo-EM structure of Formetrol-?2AR-Gs complex and cAMP function experiment to couplled the differences of receptor conformations under different ligand binding with the different downstream activation effects.It was found that different ligand efficacys were mainly attributed to the ligand-induced GPCRs conformational changes.And relevant experimental datas had been obtained to link ligand-receptor binding with nucleotide exchange process of G proteins.In this chapter,we selected long-acting ligand Formetrol,and compared with structure of BI167107-?2AR-Gs complex to establish connections between GPCRs structure and function through downstream G protein activation.Besides,we simplely discribed the 19F specific introduced method and 19F-NMR technique to analyze the chemical displacement and relaxation data of proteins in situ condition.To establish a feasible analytical method for the interaction of proteins in situ or approximate in situ conditions.We are also trying to apply this method to study the interaction between GPCRs and G proteins.