The high relevance of G protein-coupled receptors (GPCRs) becomes apparent from their diverse distribution in human tissue and their participation in a variety of physiological functions. This makes them highly interesting molecules for basic research and also a major target for pharmaceutical drugs.
Besides the structural architecture of the seven transmembrane proteins and its interaction with ligands and intracellular binding partners, their dynamic properties represent a fundamental role for their function in signal transduction. Recently, we were able to get insight into the dynamics of peptide-binding class A GPCRs by solid-state NMR spectroscopy. It was possible to characterize the dynamics of the receptors in the presence and in the absence of different ligands and embedded in different membranes [1,2,3]. The order parameters that were derived from the Dipshift experiments gave a measure for the dynamic properties of the receptors. However, they were determined for the fully 13C labeled neuropeptide Y and growth hormone secretagogue receptors, and thus represent average values of all residues. They did not resolve any specific properties of distinct sites. Here we show how to use cell-free expression for selective isotopic labelling of GPCRs. The receptors are expressed in the precipitated form and are reconstituted in lipid bicelles. The resulting NMR spectra show a reduced spectral complexity and allow the investigation of specific amino acid sites, e.g. with respect to receptor activation.
1.) Schmidt, P., Thomas, L., Müller, P., Scheidt, H. A., & Huster, D., Chemistry, 2014, 20(17), 4986-4992.
2.) Thomas, L., Kahr, J., Schmidt, P., Krug, U., Scheidt, H. A., & Huster, D. J. Biomol.- NMR, 2015, 61(3-4), 347-359.
3.) Schrottke, S., Kaiser, A., Vortmeier, G., Els-Heindl, S., Worm, D., Bosse, M., Schmidt, P., Scheidt, H.A., Beck-Sickinger, A.G., & Huster, D., Sci. Rep., 2017, 7, 46128.