Energy landscape of GPCR-mediated G-protein activation by solution-state NMR

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G-protein coupled receptors (GPCRs) are essential components of the signal transduction pathways between cells and thus are targets of around 60% of all currently marketed drugs. Despite a growing number of crystal structures, the mechanism of GPCR-mediated signaling is still not sufficiently well understood. One reason might be experimental difficulties to characterize sparsely populated activated states that occur during signaling using X-ray crystallography. In this project, we will characterize the structural and dynamical changes in a GPCR upon binding to an antagonist, agonist or G-protein using solution-state NMR spectroscopy as a main tool. In order to be able to study such a receptor system by NMR, we use a signaling-competent and stabilized GPCR that can be produced in E. coli, providing the possibility for cutting-edge isotope labeling. Our preliminary data show that this functional GPCR represents an excellent model system for NMR-based studies of the essential GPCR activation processes in a so-far unprecedented quality and resolution. In the second part of the proposed project we will study the structural and dynamical changes within a G-protein that are induced by a native membrane environment and by binding to an activated GPCR. Our membrane system of choice are phospholipid nanodiscs that are being developed for NMR applications in the lab. We will develop biochemical methods for membrane attachment of an isotope-labeled G-protein as well as for the establishment of a stable complex with a GPCR in a native-like lipid bilayer environment. These studies will provide essential information on GPCR-mediated signal transduction by investigation of the structure and functional dynamics of sparsely populated activated states occurring during the activation process in GPCRs as well as G-proteins.