Abstract
In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ?-[13CH3] labelled thermostabilized ?1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with ?1 adrenergic receptor (?1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of t?1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.
[NMR paper] Structural Biology of Human GPCR Drugs and Endogenous Ligands - Insights from NMR Spectroscopy.
Structural Biology of Human GPCR Drugs and Endogenous Ligands - Insights from NMR Spectroscopy.
Structural Biology of Human GPCR Drugs and Endogenous Ligands - Insights from NMR Spectroscopy.
Methods. 2020 Sep 07;:
Authors: Ferré G, Eddy M
Abstract
G protein-coupled receptors (GPCRs) represent the largest class of "druggable" proteins in the human genome. For more than a decade, crystal structures and, more recently, cryoEM structures of GPCR complexes have provided unprecedented insight into GPCR drug binding and cell...
nmrlearner
Journal club
0
09-13-2020 09:18 AM
[NMR paper] Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy.
Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy.
J Magn Reson. 2015 Apr;253:111-8
Authors: Kimata N, Reeves PJ, Smith SO
Abstract
G protein-coupled receptors (GPCRs) span cell membranes with seven transmembrane helices and respond to a diverse array of extracellular signals. Crystal structures of...
nmrlearner
Journal club
0
03-24-2015 09:58 PM
[NMR paper] Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy
Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy
Publication date: April 2015
Source:Journal of Magnetic Resonance, Volume 253</br>
Author(s): Naoki Kimata , Philip J. Reeves , Steven O. Smith</br>
G protein-coupled receptors (GPCRs) span cell membranes with seven transmembrane helices and respond to a diverse array of extracellular signals. Crystal structures of GPCRs have provided key insights into the architecture of these receptors and the role of conserved residues. However, the question of how ligand binding induces the...
[NMR paper] The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media.
The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media.
Related Articles The Use of Amphipols for Solution NMR Studies of Membrane Proteins: Advantages and Constraints as Compared to Other Solubilizing Media.
J Membr Biol. 2014 Mar 28;
Authors: Planchard N, Point E, Dahmane T, Giusti F, Renault M, Le Bon C, Durand G, Milon A, Guittet E, Zoonens M, Popot JL, Catoire LJ
Abstract
Solution-state nuclear magnetic resonance studies of membrane proteins are...
nmrlearner
Journal club
0
03-29-2014 01:00 PM
[NMR paper] Conformational Ensembles in GPCR Activation.
Conformational Ensembles in GPCR Activation.
Related Articles Conformational Ensembles in GPCR Activation.
Cell. 2013 Jan 31;152(3):385-6
Authors: Vardy E, Roth BL
Abstract
Recent advances in G-protein-coupled receptor structural biology have provided only limited insight into the active conformations of these key signaling molecules. A paper from Nygaard et*al. reveals the dynamic nature of GPCRs along the activation pathway by complementing NMR experiments with ultralong-timescale molecular dynamics simulations.
nmrlearner
Journal club
0
02-05-2013 09:51 PM
Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant
Mapping of unfolding states of integral helical membrane proteins by GPS-NMR and scattering techniques: TFE-induced unfolding of KcsA in DDM surfactant
September 2012
Publication year: 2012
Source:Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1818, Issue 9</br>
</br>
Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes...
nmrlearner
Journal club
0
02-03-2013 10:13 AM
[NMR paper] Studying excited states of proteins by NMR spectroscopy.
Studying excited states of proteins by NMR spectroscopy.
Related Articles Studying excited states of proteins by NMR spectroscopy.
Nat Struct Biol. 2001 Nov;8(11):932-5
Authors: Mulder FA, Mittermaier A, Hon B, Dahlquist FW, Kay LE
Protein structure is inherently dynamic, with function often predicated on excursions from low to higher energy conformations. For example, X-ray studies of a cavity mutant of T4 lysozyme, L99A, show that the cavity is sterically inaccessible to ligand, yet the protein is able to bind substituted benzenes rapidly....
GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy.
Linear Mode
