Current resolved structures of GPCRs and G protein complexes provided important insights into G protein activation. However, the binding or dissociation of GPCRs with G protein is instantaneous and highly dynamic in t...Current resolved structures of GPCRs and G protein complexes provided important insights into G protein activation. However, the binding or dissociation of GPCRs with G protein is instantaneous and highly dynamic in the intracellular environment. The conformational dynamic of G protein still needs to be addressed. In this study, we applied ^(19)F solution NMR spectroscopy to monitor the conformational changes of G protein upon interact with detergent mimicking membrane and receptor. Our results show that there are two states equilibria in the G_(α)in apo states. The interaction of G_(α)with detergents will accelerate this conformational transformation and induce a state that tends to bind to GPCRs. Finally, the G_(α)proteins presented a fully activation state when they coupled to GPCRs.展开更多
Cyclic peptides are an attractive class of bioactive molecules whose proven utility in the research lab and clinic has fueled the development of new candidates with diverse chemical structures and functions.However,th...Cyclic peptides are an attractive class of bioactive molecules whose proven utility in the research lab and clinic has fueled the development of new candidates with diverse chemical structures and functions.However,these candidates have been largely limited to naturally evolved products or their derivatives.Herein,we describe the de novo discovery of bicyclic peptides using a phage-encoded library of peptides possessing the characteristic framework of naturally occurringα-conotoxins(α-Ctxs).Selection againstα7 nicotinic acetylcholine receptor yielded a bicyclic peptide possessing an uncanonical disulfide connectivity rarely found in naturally occurringα-Ctxs.The chemically synthesized analogue of the hit demonstrated a more potent inhibitory effect on the receptor(IC_(50)=8.6 nmol/L)compared to the majority of known naturalα-Ctxs.Cryo-EM structural studies revealed that the binding mode of this selected peptide to the receptor differed from that of naturalα-Ctxs,based on the orientation of the peptide in the binding pocket and its contacts with the receptor.Our work highlights the utility of a natural toxin-based directed evolution strategy to identify conformationally constrained peptide binders of target proteins,and is expected to accelerate the discovery of well-behaved peptides for use as biological probes and therapeutics.展开更多
Dear Editor,The functional diversity of proteins is related to the cooperation of multiple domains.Independent globular domains are typically joined by a fl exible length of polypeptide chain,which makes the structura...Dear Editor,The functional diversity of proteins is related to the cooperation of multiple domains.Independent globular domains are typically joined by a fl exible length of polypeptide chain,which makes the structural analysis of multi-domain proteins diffi cult.Here,we describe the combined use of solution NMR(nuclear magnetic resonance)and EPR(elec-tron paramagnetic resonance)for the structural analysis of a protein with two separate domains.The structure of each domain was determined independently using conventional NMR restraints,and the relative orientation of the two separate domains was confi ned using long-distance restraints obtained by NMR-PRE(paramagnetic relaxation enhancement)and EPR-DEER(double electron-electron resonance,also called PELDOR:pulsed electron double reso-nance.展开更多
基金supported by the National Key Research and Development Project of China (Nos.2019YFA0904100 and 2017YFA0505400)the National Natural Science Foundation of China (Nos.22077117 and 31971152)the USTC Research Funds of the Double First-Class Initiative。
文摘Current resolved structures of GPCRs and G protein complexes provided important insights into G protein activation. However, the binding or dissociation of GPCRs with G protein is instantaneous and highly dynamic in the intracellular environment. The conformational dynamic of G protein still needs to be addressed. In this study, we applied ^(19)F solution NMR spectroscopy to monitor the conformational changes of G protein upon interact with detergent mimicking membrane and receptor. Our results show that there are two states equilibria in the G_(α)in apo states. The interaction of G_(α)with detergents will accelerate this conformational transformation and induce a state that tends to bind to GPCRs. Finally, the G_(α)proteins presented a fully activation state when they coupled to GPCRs.
基金supported by the Science and Technological Fund of Anhui Province for Outstanding Youth(2208085J21)the National Natural Science Foundation of China(22227810,21825703,22137005,and 92253302)the Beijing Life Science Academy Scientific Research Program(2023000CA0070)。
文摘Cyclic peptides are an attractive class of bioactive molecules whose proven utility in the research lab and clinic has fueled the development of new candidates with diverse chemical structures and functions.However,these candidates have been largely limited to naturally evolved products or their derivatives.Herein,we describe the de novo discovery of bicyclic peptides using a phage-encoded library of peptides possessing the characteristic framework of naturally occurringα-conotoxins(α-Ctxs).Selection againstα7 nicotinic acetylcholine receptor yielded a bicyclic peptide possessing an uncanonical disulfide connectivity rarely found in naturally occurringα-Ctxs.The chemically synthesized analogue of the hit demonstrated a more potent inhibitory effect on the receptor(IC_(50)=8.6 nmol/L)compared to the majority of known naturalα-Ctxs.Cryo-EM structural studies revealed that the binding mode of this selected peptide to the receptor differed from that of naturalα-Ctxs,based on the orientation of the peptide in the binding pocket and its contacts with the receptor.Our work highlights the utility of a natural toxin-based directed evolution strategy to identify conformationally constrained peptide binders of target proteins,and is expected to accelerate the discovery of well-behaved peptides for use as biological probes and therapeutics.
基金This research was supported by the National Basic Research Program(973 Program)(Nos.2011CB911104 and 2013CB910200)the Chinese Natural Science Foundation of China(Grant No.31100563)to Y.Xiong and(Grant No.31170817)to C.Tian.
文摘Dear Editor,The functional diversity of proteins is related to the cooperation of multiple domains.Independent globular domains are typically joined by a fl exible length of polypeptide chain,which makes the structural analysis of multi-domain proteins diffi cult.Here,we describe the combined use of solution NMR(nuclear magnetic resonance)and EPR(elec-tron paramagnetic resonance)for the structural analysis of a protein with two separate domains.The structure of each domain was determined independently using conventional NMR restraints,and the relative orientation of the two separate domains was confi ned using long-distance restraints obtained by NMR-PRE(paramagnetic relaxation enhancement)and EPR-DEER(double electron-electron resonance,also called PELDOR:pulsed electron double reso-nance.
