New Polymer Belts for Lipid-Bilayer Nanodiscs to Study the Molecular Basis of GPCR Signalling
Biophysics
Final Report Abstract
Our general aim is to understand how G protein-coupled receptors (GPCRs) rearrange overtime when activated by their ligands, which means studying their structural dynamics. This is an exciting biological question because not only structural changes but also the molecular dynamics of GPCR activation determine their pharmacology. Specifically, we aim at developing fluorescence-based sensors to track the relative movements of single receptor domains. In this context, polymer nanodiscs would offer the possibility of investigating the receptors in vitro – with a strict control of experimental conditions – yet in an environment that is very close to the natural membrane. We apply genetic code expansion technology to incorporate non-canonical amino acids for bioorthogonal chemistry into the GPCRs. Using these chemical anchors, we can label the receptors at any solvent-accessible position with desired fluorophores, both in vitro and on the surface of live cells. While working on incorporating two labels into our GPCRs to build FRET reporters, we serendipitously discovered that even a single fluorophore installed at specific GPCR sites can change its emission intensity upon receptor activation, probably due to a change in the local environment (polar/nonpolar) around the chromophore. Building on this discovery, we established a new class of GPCR biosensors that allow the tracking of conformational rearrangements at a single-residue level directly on the surface of live cells. Using the muscarinic M2 acetylcholine receptor as a model for a GPCR endogenously activated by a small-molecule ligand, we show that pharmacologically distinct agonists stabilize different receptor conformations. Moreover, by analyzing the kinetics of the fluorescence changes in different regions of the receptor, we demonstrate that activation does not occur through a concerted movement of the entire receptor but rather follows a sequence of sequential conformational changes in individual domains. By applying the same type of sensors to a rhodopsin-like GPCR that naturally binds large peptide ligands, the Y5 receptor, we show that the 7-transmembrane (7TM) core rearranges faster than the soluble N-terminus (NT) during peptide binding. Although we know from crosslinking data that the NT forms extensive contacts with the ligand, the kinetics suggest that the NT is not responsible for peptide recruitment, which is the case for secretin-like GPCRs. Furthermore, we show that G protein coupling affects only the kinetics of the receptor core and that the core is responsible for ligand selectivity. Overall, we have established a new system to track GPCR conformational rearrangements without extracting them from their natural cellular environment. This technique can, in principle, be applied to any GPCR. We are currently working on transferring the methodology in vitro and using smaller fluorophores for more precise tracking of backbone movements.
Publications
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Noncanonical Amino Acid Tools and Their Application to Membrane Protein Studies. Chemical Reviews, 124(22), 12498-12550.
De Faveri, Chiara; Mattheisen, Jordan M.; Sakmar, Thomas P. & Coin, Irene
