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Title: The development of solid-state NMR methodology to study the dynamics of proteins and ice
Author: Stevens, Rebecca A.
ISNI:       0000 0004 7972 4350
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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Solid-state nuclear magnetic resonance (SSNMR) is an excellent tool for determining the molecular motions within a dynamic system. SSNMR relaxation measurements can access a vast range of timescales (ps - ms) and are able to simultaneously determine the frequency and amplitude of the motion that a particular nucleus is undergoing. Recent developments in SSNMR instrumentation now allow for >100 kHz magic angle spinning (MAS) using 0.7 and 0.8 mm rotors. State-of-the-art MAS is especially beneficial for those wishing to investigate proteins in the solid state: only sub-milligram amounts of sample are required and the fast spinning yields incredible spectral resolution. This also enables proton detection and the associated improvements in sensitivity (for protonated samples). Unfortunately, these small rotors are extremely challenging to pack with the semi-solid protein samples. Furthermore, the proteins can become dehydrated in the slow packing process, making them unsuitable for NMR. To address this point, in Chapter 3, we present the design and application of an ultracentrifuge tool for the packing of proteins into 0.7 - 1.3 mm diameter SSNMR rotors. The tool helps to reduce the waste of expensive isotopically labelled proteins and decreases the packing time from several hours to minutes. The work in Chapter 4 takes advantage of the mentioned fast MAS developments and demonstrates the accurate measurement of site-specific, spin-lattice relaxation rates (R1) on 13Ca nuclei in a fully protonated, uniformly 13C-labelled protein at 100 kHz MAS. Our approach overcomes the averaging effect of proton-driven spin diffusion that obscures site-specific information for the relaxation rates measured at slower spinning frequencies. One area where measurements of relaxation in the solid state can yield significant insights is the understanding of the complex energy landscape describing conformational changes of proteins, which are often closely linked to their functions. In Chapter 5 we present some of the first extensive site-specific variable temperature measurements of 13C' and 15N R1 and spin-lattice relaxation rates in the rotating frame (R1r) in a crystalline protein as a way to explore its conformational energy landscape. We observe R1r more than doubling over a narrow range of temperatures and minimal variation in R1 over the same range. We model the relaxation data using an extended model free approach and Arrhenius relationship to extract activation energies for the motions dominating the dynamics, however _nd that further measurements are required for an accurate determination of the activation energies. In Chapter 6 we show that relaxation measurements in the solid state are not only useful for characterising protein motions. In this chapter, we employ variable temperature measurements, including relaxation measurements, to investigate the effects of non-colligative antifreezes on ice dynamics. Antifreeze (glyco)proteins facilitate the survival of a diverse range of organisms at low temperatures by altering the freezing point, structure and growth of ice by modifying the dynamics of water molecules. These proteins and their synthetic mimics have many vital applications throughout science and engineering, but their mechanism of action is still not completely understood. In this PhD project, a combination of variable temperature relaxation measurements and 2D exchange spectra revealed that the antifreeze glycoproteins, type I antifreeze proteins, safranin and polyvinyl alcohols were exploiting a similar antifreeze mechanism involving reversible binding to ice, whereas the type III antifreeze protein was irreversibly binding to ice.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry