Theses and Dissertations
Permanent link for this collectionhttps://hdl.handle.net/2022/3086
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Browsing Theses and Dissertations by Subject "19F"
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Item ANALYSIS OF NEUROTENSIN RECEPTOR 1 CONFORMATIONAL DYNAMICS AND ALLOSTERIC INFLUENCES IN ACTIVATION(2022-10) Dixon, Austin D.; Ziarek, Joshua J.G protein-coupled receptors (GPCRs) are the largest integral membrane protein class in eukaryotes with over 800 unique members that regulate numerous biological processes including mood, body temperature, taste, and sight, amongst others. Due to their broad physiological importance and numerous etiological roles, GPCRs are the targets for more than 30% of all therapeutic drugs on the market. A more nuanced mechanistic understanding of the GPCR activation landscape could dramatically expand their therapeutic value. Unfortunately, accurately and efficiently assessing GPCR activation and cognate transducer stimulation is difficult due to inherently poor protein stability and expression; this is further hindered by challenging protein purification requirements. This dissertation seeks to expand the knowledge of GPCR conformational dynamics and their connection to receptor activation and ternary complex formation with transducer molecules G protein and Arrestin. Neurotensin receptor 1 (NTS1) has quickly become one of the most well-characterized GPCRs with structures of the apostate, complexes with various pharmacological ligands, and ternary complexes with both the heterotrimeric Gi protein and β-arrestin-1 (βArr1) transducers. NTS1 is a class A, β group receptor that is expressed throughout the central nervous system and the gastrointestinal (GI) tract. Activation by its endogenous tridecapeptide ligand neurotensin (NT) mediates a variety of physiological processes including low blood pressure, high blood sugar, low body temperature, mood, and GI motility. It is also a long-standing therapeutic target for Parkinson’s disease, schizophrenia, obesity, hypotension, psychostimulant substance use disorders, and cancer. Using NTS1 as a model GPCR, this dissertation details the development of a novel method for Selective 19F-Labeling Absent of Probe Sequestration (SLAPS), which allows for accurate and efficient assessment of GPCR activation by ligands and transducer complexation via solution nuclear magnetic resonance (NMR) spectroscopy. Through 19F NMR and other biophysical techniques, this dissertation shows that the NTS1 allosteric activation mechanism may be alternatively dominated by induced fit or conformational selection depending on the coupled transducer, and the available static structures do not represent the entire conformational ensemble observed in solution. Furthermore, this dissertation explores the pleiotropic effects of biased allosteric modulators and lipids in NTS1 activation and ternary complex formation.