Advances in Sensitivity and Resolution of Solid State Nuclear Magnetic Resonance and Dynamic Nuclear Polarization

Abstract

Traditional structural biology methods such as x-ray crystallography and solution-state nuclear magnetic resonance (NMR) cannot provide atomic level resolution of insoluble, noncrystalline proteins. Such proteins include amyloid fibrils implicated in numerous neurodegenerative diseases. While solid state NMR using magic angle spinning (MAS) has yielded high resolution structures of amyloid fibrils, including polymorphs of amyloid-β (Aβ), its sensitivity is inherently limited, requiring undesirable time and resource commitments. Dynamic nuclear polarization (DNP) is a powerful method of enhancing solid state NMR sensitivity. However, this sensitivity gain comes at a significant loss of spectral resolution due to sample conformational heterogeneity at the cryogenic temperatures required for efficient electron-nuclear polarization transfers. This hinders acquisition of site-specific structural assignments in complex systems. Thus, it is critical to improve DNP resolution by advancing to higher magnetic fields and faster MAS frequencies. Unfortunately, at high fields, the efficiency of the most widely applied DNP polarization mechanisms decrease, along with the availability of high-power microwave sources. The work presented in this thesis seeks to address instrumentation limitations and improve DNP methods that presently limit the utility and power of MAS DNP at high fields.

This thesis first describes the mechanism of Overhauser Effect (OE) sensitivity enhancements in insulating solids. We demonstrate the generation of strong positive OE with less than 200 mW of microwave power. We also employ selective deuteration to elucidate the role of individual hyperfine-coupled protons on the BDPA radical. This work provides a basis for the improved development of high field DNP radicals with fluctuating hyperfine interactions.

The continued expansion of MAS DNP at high field and with fast MAS rotors requires improvement in the efficiency of coupling microwave irradiation into the sample. We provide a comprehensive discussion of the effect of the radio frequency (RF) coil on the transverse microwave coupling efficiency in 1.3 mm and 0.7 mm rotor systems. When the ratio of the pitch to microwave wavelength is ~ 0.5, the coupling efficiency is significantly reduced, as is the case for a typical 1.3 mm or 0.7 mm RF coil at fields between 460-593 GHz. To address this, we introduce axial microwave coupling schemes for 3.2, 1.3-, and 0.7-mm rotors and demonstrate theoretical improvements in the electron Rabi field of > 60% in 3.2 mm rotor systems and up to a factor of 8 improvement in 0.7 mm rotor systems. We further provide experimental results of MAS spinning stability in 3.2 mm rotors at 95 K using the modified axial bearing required for axial irradiation schemes.

While the first two sections describe sensitivity enhancements under DNP, the later chapters are focused on sensitivity enhancements leveraged via MAS frequencies > 90 kHz and ¹H detected MAS NMR. We demonstrate the first ¹H detected MAS NMR study of the arctic mutant of Aβ₁₋₄₂, which is implicated in the pathogenesis of early onset familial AD. Despite resolution limitations in the sample as a result of limited MAS frequency and sample heterogeneity, we determine that the core fibril structure of E22G Aβ₁₋₄₂ is monomorphic with suggested conserved structure relative to that of the wild type fibril.

To further reduce homogenous contributions to the solid-state linewidth, we introduce the fabrication and use of 0.7 mm diameter diamond rotors. The superior material strength, thermal conductivity, and microwave transparency make diamond the optimal MAS rotor material. First, we describe the mechanism of material ablation and characterize the effects of pulse energy, irradiation scheme and pulse number on the achievable taper angle in high aspect ratio holes. We then apply a dual-sided axial machining strategy to fabricate 0.7 mm diamond rotors, and further demonstrate stable operation up to 124 kHz in addition to ¹H detected MAS NMR results. Overall, the areas of focus in this thesis describe several resolution and sensitivity advancements that when combined in the future could provide sufficient sensitivity and resolution with which to study ex-vivo amyloid plaque samples and other exogenous biomedically relevant samples.