Characterization of protein structure and dynamics by solution and solid-state NMR

Abstract

The study of proteins for understanding biological processes or as an approach to treating diseases has become more relevant in recent years. Different structural biology approaches can help to answer some of these questions, including nuclear magnetic resonance (NMR) spectroscopy, which provides information at atomic resolution about the structure and dynamics on a broad range of timescales. Traditionally, protein studies are carried out by solution NMR, where many methodologies have been developed over the years that allow one to obtain a great variety of information. However, in solution NMR studies, the protein size is limited to up to 50 kDa. Using different labeling schemes and different levels of protonation in the protein, with solid-state NMR and 1H detection, we can access biologically relevant systems with a molecular size larger than 50 kDa and extract information of their structure and dynamics on a different timescale with atomic resolution. This thesis presents a combination of solution and solid-state NMR methodologies to understand the dynamical processes of the MAP kinase p38α and their changes upon ligand binding as a tool for characterizing the allosteric dynamic network present in the protein. All this was done by combining different labeling schemes and proving new methodologies for acquiring relaxation dispersion experiments that can be used for large protein systems. Furthermore, relaxation dispersion techniques in solid-state NMR are used to characterize the modulation of dynamics by inter- and intramolecular interaction for the SH3 domain of chicken α-spectrin by introducing a mutation in the RT loop. Furthermore, this work presents the characterization of the secondary structural element for the N-terminal domain of RhoGDI1, a domain thought to be disordered in the apo form and ordered in complex with the GTPase client. However, with the experimental chemical-shift information, we proved it to have a secondary structure before complex formation.