The Research Of The Activation Mechanism Of GABA_B Receptor Based On The Active-Based Probes

G protein-coupled receptors （GPCRs） mediate physiological responses to variousligands, such as hormones, neurotransmitters and sensory stimuli. We synthesized bothactive and affinity tagged photo-affinity probes specific to GABA_Breceptor. Suchactivity-based probes enabled us to first lock GABA_Breceptor in an inactive state and thenstimulate the receptor with a positive allosteric modulator, thereby permitting monitoringof the dynamic events associated with GABA_Breceptor activation. We found thatactivation of GABA_Breceptor triggered a dynamic assembly and disassembly of a proteincomplex, including GABA_Breceptor, IGF-1R and its downstream effectors. Notably, bothGαi and Gβ s ubunits were pre-assembled with the inactive GABA_Breceptor anddisassembled from the receptor complex upon activation. Our results identify a criticalrole for activity-specified dynamic protein interactions between GABA_Breceptor anddownstream signaling proteins.The extracellular domain of GABA_B1subunit (GABA_B1-VFT) contains the Ligandbinding pocket （LBP） and was an excellent therapeutic target. For the further researches ofthe GABA_B1-VFT domain structure, the recombinant GABA_B1-VFT was cloned,expressed and purified in large scale. The functional fraction of GABA_B1-VFT wasisolated by the ligand affinity chromatography. This fraction has the high activity ofligand binding.In order to study the process of GABA_Breceptor ligand bind, we designed a platformconstructed by ESI-MS, Circular Dichroism Spectroscopy and Fluorescence Spectroscopywhich can monitor the Secondary structure and the Tertiary structure changes as well asthe ligands binding or dissociation process. With this platform, we analyzed the Mbconformational changes during its ligand binding or dissociation process in differentconditions, and demonstrated the different mechanism of the heme releasing induced byacid and organic solvents.