Research On The Dynamic Process Of G Protein And G-protein Coupled Receptors Based On ¹⁹F NMR Technology

G protein-coupled receptors(GPCRs)are the largest family of membrane protein receptors.Because they participate in almost all physiological processes in the human body,they have become a key target for drug development.Therefore,a detailed understanding of the structure and function of GPCRs is of great value for the development of new treatment methods and the design of synthetic receptors and biosensors.The high-resolution complex structure analyzed by X-ray and Cryo-EM provides a structural basis for the development of drugs that selectively target GPCRs downstream signals.However,the static crystal structure and cryo-electron microscope structure cannot provide sufficient information about the dynamic process of G protein coupling to GPCRs.Nuclear Magnetic Resonance Spectroscopy(NMR)is one of the key methods for studying dynamic characteristics of proteins and protein complexes at physiological temperatures.Compared with other widely used NMR probes(such as 1H,13C,15N or 31P),stable NMR active nuclear fluorine(19F)is almost non-existent in biological systems,which makes it a unique organism Orthogonal atoms are used to detect molecular interactions in biology.In addition,the 19F probe is very sensitive to the chemical environment changes,resulting great advantages in studying the dynamics of membrane proteins like GPCRs.The combination of G protein and membrane plays a vital role in the function of G protein,although there have been a lot of studies on the interaction between polypeptide or protein and plasma membrane from the aspects of site-directed mutagenesis,biochemistry and function.Inquiry,due to the transient and extremely dynamic interactions,the research on G protein-membrane interactions is still limited.Therefore,we tried to apply 19F NMR technology to the study of G protein-plasma membrane and the structural dynamics of G protein-β2AR in order to better understand the molecular mechanism of the GPCRs-mediated G protein activation.In the first part,we site-specificly introduced the 4-trifluoromethyl-phenylalanine(tfmF)into the interaction interface of GPCR and Gα protein,and explored the Gα protein changes in the conformational state of apo and the interaction with the mimicking membrane with high sensitive 19F-NMR detection.Our results show that there are two states equilibria in the Ga in apo states and the interaction of Gα with membrane will accelerate this conformational transformation.In the second part,the conformational changes of Gα proteins(Gαs and Gαi)binding on the activated GPCR was investigated by 19F NMR detection.Taking the typical GPCR biological model β2 adrenergic receptor(β2AR)as the research object,we expressed the β2AR receptor used the Bac-to-Bac insect baculovirus expression system and purified the activated receptor using affinity chromatography.A certain concentration of Gα protein was titrated with a known concentration of activatedβ2AR receptor to monitor the interaction process of β2AR receptor-Gα.The results of one-dimensional 19F NMR showed that the conformation of Gαs and Gαi proteins was further stabilized by coupling with β2AR.At the same time,we analyzed the differences in the interaction of Gαi,Gαs and β2AR,and concluded that β2AR can be coupled to either Gαs or Gαi,and may tend to couple to Gαs.These differences observed from the one-dimensional NMR spectrum are consistent with the existing research results,proving that 19F NMR technology can be used as a convenient and effective tool to study the dynamic process of the GPCR-G protein signaling pathway.