Independent Activation Mechanism Of Arrestin With Transmembrane Helices Of GPCRs Reavealed By Genetically Encoded Trimethylsilyl Hydrogen Nuclear Magnetic Probe (TMSiPhe)

In the world of biomolecules,proteins are not alone,the functions of which were realized to rely on working in specific three-dimensional conformation and with some other molecules(small molecule ligands,proteins,nucleic acids,etc.).The interaction between protein and protein is the main activities of the cell biochemical reaction network,the characterizations of which has important significance for understanding physiological and pathological processes,and designing drug molecules.As the largest family of membrane receptors,by working with extracellular signaling molecules,and coupling with intracellular G proteins or Arrestin proteins,G protein-coupled receptors(GPCRs)transmit chemical signals into the cells to regulate many physiological processes.Based on this,more than one-third of drugs in markets are targeted to GPCRs.In a word,the structural characterization of GPCRs signal complexes is the basis for understanding many physiological processes and plays a key role in drug development.Although the rapid development in technologies of X-ray crystallography and cryo-electron microscopy have made structural characterization of these complexes possible in recent years,these methods rely on the stability of the complexes,and usually,only the structural details of the rigid part are present in high resolution.As a powerful tool for the structure solution and dynamic detection for soluble proteins,nuclear magnetic resonance(NMR)can detect key signals on the formation of transient complexes,especially the dynamic structural information of the flexible regions of the complex,which is hard to obtain for other structural analysis methods.However,for the traditional NMR experiments,the assignment of signal peaks of protein with larger molecular weights is complicated and difficult,sample amount and acquire time are both considerable.NMR is still difficult to be widely applied in the study of GPCRs signal complexes.The deficiency in complex structure and dynamic information has become a gap for drug design to GPCRs(Rational Drug Design,for example,ligands designed to bias Arrestin protein function)).Here we established a new strategy in this study:specifically incorporated 4-trimethylsilylphenylalanine(TMSiphe)into specific sites of Arrestin protein in E.coli using genetic encoded unnatural amino acid(UAA)tecnology.The conformational change of the GPCRs-Arrestin signal complex was detected by using the trimethylsilyl group as a nuclear magnetic resonance probe.Unique nuclear magnetic resonance chemical shift and high-intensity resonance absorption signal of TMSiphe make it easy to be identified at very low concentrations(5?10?M),and the acquire time is shortened to a few minutes.Moreover,the probe is quite sensitive to structural changes,which can detect the active state of the protein and determine the binding affinity between proteins.In this study the direct regulation of Arrestin protein conformation by the receptor transmembrane core(TM Core)region was detected in the ligand-GPCRs-Arrestin complex,suggesting that it is possible to design of the Arrestin-biased ligand and avoiding endogenous biased pathways(eg differences in GRK expression in different cell environments).Our newly developed strategy has unique advantages in the study of conformational changes in GPCRs complexes,which provides a structural basis for understanding the function of GPCRs and designing biased drugs.With the improvement of the technologies on recombinant expression and purification of GPCRs proteins and complexes,simulation of cell membrane environmental,screening unnatural amino acid screening and NMR pulse sequences,TMSiPhe will have broader application as a nuclear magnetic probe.Additionally,It is striking that this is the first time for organosilicon compounds to be incorporated in the proteins in cells.The structural solution of the corresponding aminoacyl tRNA synthetase reveals key structural information and recognition sites,which is important for the study of artificial evolution of enzymes and research on silicon-based life.