NMR spectroscopy of disordered and multidomain proteins: methodological and structural insights

Nuclear magnetic resonance (NMR) spectroscopy is a well-established technique to investigate the structure, dynamics, and interactions of biomolecules, such as proteins and nucleic acids. Application of NMR is particularly valuable for characterizing intrinsically disordered proteins (IDPs), a class of proteins that lack a stable 3D structure and possesses high flexibility. Disorder can also be found within multidomain proteins, in the form of intrinsically disordered regions (IDRs). Many of these modular proteins are composed of well-folded globular domain separated by IDRs of various length, that until recently were thought to function merely as linkers. However, many studies have shown that these disordered regions play significant roles ranging from mediating interactions with partners to modulating protein function. Moreover, both IDPs and multidomain proteins have important biological functions and their disfunction can lead to the onset of many pathologies, such as cancer and neurodegenerative diseases. Therefore, studying these systems is crucial to understanding their molecular mechanisms of action, and NMR can significantly contribute to this goal by providing high-resolution data. However, further advancements are required to push the boundaries of state-of-the-art NMR techniques for studying this important class of biologically relevant proteins. In this thesis, the contribution to the advancement of NMR spectroscopy for the study of challenging disordered systems is described. The newly developed methodologies enable the characterization of multidomain proteins, the acquisition of high-quality spectra of large IDPs at ultra-high magnetic field, and the application of paramagnetic NMR to disordered systems. Furthermore, NMR spectroscopy was applied to investigate biologically relevant IDPs and their interactions.