Cross polarization stability in multidimensional NMR spectroscopy of biological solids.

Magic-angle spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy is a powerful and versatile technique for probing structure and dynamics in large, insoluble biological systems at atomic resolution. With many recent advances in instrumentation and polarization methods, technology development in SSNMR remains an active area of research and presents opportunities to further improve data collection, processing, and analysis of samples with low sensitivity and complex tertiary and quaternary structures. SSNMR spectra are often collected as multidimensional data, requiring stable experimental conditions to minimize signal fluctuations (t1 noise). In this work, we examine the factors adversely affecting signal stability as well as strategies used to mitigate them, considering laboratory environmental requirements, configuration of amplifiers, and pulse sequence parameter selection. We show that Thermopad® temperature variable attenuators (TVAs) can partially compensate for the changes in amplifier output power as a function of temperature and thereby ameliorate one significant source of instability for some spectrometers and pulse sequences. We also consider the selection of tangent ramped cross polarization (CP) waveform shapes, to balance the requirements of sensitivity and instrumental stability. These findings collectively enable improved stability and overall performance for CP-based multidimensional spectra of microcrystalline, membrane, and fibrous proteins performed at multiple magnetic field strengths.

Solid-State NMR 13C sensitivity at high magnetic field.

Sensitivity is the foundation of every NMR experiment, and the signal-to-noise ratio (SNR) should increase with static (B0) magnetic field, by a proportionality that primarily depends on the design of the NMR probe and receiver. In the low B0 field limit, where the coil geometry is much smaller than the wavelength of the NMR frequency, SNR can increase in proportion to B0 to the power 7/4. For modern magic-angle spinning (MAS) probes, this approximation holds for rotor sizes up to 3.2 mm at 14.1 Tesla (T), corresponding to 600 MHz 1H and 151 MHz 13C Larmor frequencies. To obtain the anticipated benefit of larger coils and/or higher B0 fields requires a quantitative understanding of the contributions to SNR, utilizing standard samples and protocols that reproduce SNR measurements with high accuracy and precision. Here, we present such a systematic and comprehensive study of 13C SNR under MAS over the range of 14.1 to 21.1 T. We evaluate a range of probe designs utilizing 1.6, 2.5 and 3.2 mm rotors, including 24 different sets of measurements on 17 probe configurations using five spectrometers. We utilize N-acetyl valine as the primary standard and compare and contrast with other commonly used standard samples (adamantane, glycine, hexamethylbenzene, and 3-methylglutaric acid). These robust approaches and standard operating procedures provide an improved understanding of the contributions from probe efficiency, receiver noise figure, and B0 dependence in a range of custom-designed and commercially available probes. We find that the optimal raw SNR is obtained with balanced 3.2 mm design at 17.6 T, that the best mass-limited SNR is achieved with a balanced 1.6 mm design at 21.1 T, and that the raw SNR at 21.1 T reaches diminishing returns with rotors larger than 2.5 mm.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.