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Characterisation of the conformational landscape of the β1-adrenergic receptor and development of a strategy towards NMR studies of the protease-activated receptor 1 10.17863/CAM.50235 Frei, Jan Niclas Nietlispach, Daniel The research conducted for this thesis was aimed at providing a comprehensive description of the dynamic and conformational landscape of the minimally thermostabilised avian β1-adrenergic receptor (tβ1AR) using solution state nuclear magnetic resonance (NMR) spectroscopy. The tβ1AR is a class A G-protein-coupled receptor (GPCR) and the human counterpart is an important drug target in diseases of the heart. In chapter 2, a wide range of constructs, labelling reagents and conditions were tested to make the tβ1AR accessible to 19F NMR spectroscopy. The conditions optimised in chapter 2 were used in chapter 3 to conduct comprehensive 19F NMR studies investigating the conformational plasticity of the cytoplasmic ends of transmembrane domains 6 and 7. Both domains have previously been proposed to be important structural hotspots for conformational changes during the activation of the receptor. The work described in chapter 3 tests the conformational response of both domains in response to a range of ligands and a Gs protein mimicking nanobody. The data obtained suggested that the cytoplasmic ends of the receptor sample through several inactive, pre-active and signalling active states and the populations of these states are tightly controlled by the Gs efficacies of ligands and the binding of intracellular binding partners. The results will help to explain how extracellular signals are conformationally transmitted through the receptor to the cytoplasmic face of the receptor. In chapter 4, I complemented the results obtained in the NMR spectroscopic studies of chapter 3 with structural data obtained through second harmonic generation (SHG) spectroscopy. In a final step in chapter 5, the knowledge gained from working with the tβ1AR has been applied to initiate an investigation of the conformational plasticity of a second GPCR target, the human Protease activated receptor 1 (PAR1). The PAR1 is mostly associated with regulation of haemostasis and as such a high potential target for clinical research. I established the construct, expression, purification and 19F labelling of the PAR1 and obtained initial 19F NMR spectra. The work is an important step towards understanding the PAR1 conformational landscape. University of Cambridge 2020-04-25 Thesis Doctoral Doctor of Philosophy (PhD) en Herchel Smith Fund https://www.repository.cam.ac.uk/handle/1810/303160 controlled.access https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/76cd5b97-d0b5-4406-aeb3-ceb6d9123d25/download 17064349506d1d799a4d89400a12d639 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2c562c35-64a0-459f-a4d6-47eda75099ed/download 87eda9de84448d1f82354d60eee3eb5f https://www.rioxx.net/licenses/all-rights-reserved/ G protein-coupled receptor Nuclear magnetic resonance spectroscopy β1-adrenergic receptor Protease-activated receptor 1 Second harmonic generation