New Tools for Structural Biology and Biophysics: High-Throughput Fluorine Solid-State NMR and Applications to Membrane Proteins

Solid-State Nuclear Magnetic Resonance (SSNMR) spectroscopy is a powerful method for characterizing the structure and dynamics of crystalline and amorphous solid compounds, materials, and biological systems. When applied to biomolecular systems such as membrane proteins, it can provide access to information about structure and dynamics in native environments, on targets that are difficult to characterize by other biophysical methods. Membrane proteins in particular are critical for biological function and are overrepresented as drug targets; however, they are notoriously difficult to study. In this thesis, new SSNMR methods are developed, utilizing fast Magic Angle Spinning (MAS), multidimensional correlation, and the 19F nucleus as a biophysical probe for the understanding the structure and dynamics of crystalline and membrane-bound proteins and protein-ligand complexes. Internuclear distances are critical in biomolecular structure determination. The 19F nucleus, due to its high gyromagnetic ratio, absence of natural background, small atomic radius, and highly developed chemistry is uniquely suited as a probe for measuring long internuclear distances. Utilizing uniform 13C labeling and multidimensional correlation, an experiment for multiplex measurement of 13C-19F distances is developed. Furthermore, 13C-19F coherence transfer methods are compared and optimized to enable direct 13C-19F correlation to disambiguate constraints in polyfluorinated systems. These technological developments are applied toward determining the structure of the Envelope (E) protein of the novel SARS CoV-2 virus. With fast MAS, proton-detected experiments in SSNMR are possible with high resolution and sensitivity. A new method for measuring nanometer-length distances in a multiplex manner is developed, utilizing 1H-19F Rotational Echo Double Resonance (REDOR) and two-dimensional 1H-15N correlation. The experiment is developed on a quad-labeled (uniform 2H, 13C, 15N, and 19F-tagged) model protein, and the distances measured are shown to be in quantitative agreement with the known structure. This technology is applied to refine the structure of the E. coli. multidrug resistance protein E (EmrE), by measuring a large number of 1H-19F distances between a tetrafluorinated ligand and the protein HN atoms. The structure of the EmrE protein was determined at high and low pH, modeling functional states of the transporter, and providing insight into the mechanism of the proton-coupled antiport.Ph.D