From Intrinsically Disordered Peptides And Proteins To Globular Proteins To Supramolecular Complexes: Development Of New 19F-NMR Methodologies For Increased Versatility In Characterization Of Polypeptide Biophysics, Membrane Binding, And Intermolecular Interactions

Abstract

19F-NMR spectroscopy is a powerful tool to advance our understanding of protein interactions due to a lack of background 19F signals, the inherently high sensitivity of the 19F nucleus, and minimal perturbation of proteins through 19F-labeling. In this dissertation, I explored different 19F-NMR methodologies to characterize these phenomena, including polypeptide biophysical properties, membrane binding properties and intermolecular interactions. This research encompasses two main studies that use the 19F-NMR: first, the development of bicelle-optimized 19F-NMR experiments allowing me to experimentally demonstrate proof of a novel binding mode for the bioactive peptide apelin-17 towards its cognate G-protein-coupled receptor (GPCR, the apelin receptor, AR); second, the creation and evaluation of a novel 19F-NTA-probe targeting His-tagged proteins, enhancing the specificity and utility of 19F-NMR in protein characterization. By expanding our toolbox of 19F-NMR methodologies, the findings from my study of apelin-17 binding mechanisms provided new insight into its interaction dynamics with membranes, allowing me to provide experimental support for a mechanism of GPCR binding that involves both enhanced diffusion via membrane-catalyzed peptide binding and localization combined with the intrinsically disordered fly-casting properties of its C-terminal tail – enabled through the binding mode – to enhance receptor recognition. This model not only sheds light on apelin's therapeutic potential in metabolic and neurological diseases but is also likely to have broader implications for similar peptide-GPCR systems. Furthermore, by developing a novel noncovalent 19F-probe, we characterized a 19F-labelling approach that can bypass the laborous purification procedure of 19F-labelled proteins. This 19F-probe can be applicable for probing proteins intermolecular interaction and with a promising potential for on-cell NMR studies.