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This primer article introduces a practical guide for setting up 1H detection solid-state NMR experiments under fast MAS conditions for small molecules. Sample independent adjustment/calibration including magic angle adjustment, shimming, radio-frequency field calibration, Hartmann-Hahn matching adjustments, and 1H heteronuclear decoupling are demonstrated. Experimental setups which depend on samples are also shown for 1H NMR, 1H-1H homonuclear correlation measurements, and 1H-X heteronuclear correlation/distance measurements. Information obtained from each experiment is also highlighted.

Several publications describing high-resolution structures of amyloid-ß (Aß) and other fibrils have demonstrated that magicangle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments--namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive twodimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aß1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aß1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ~6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aß and other amyloid systems. [ABSTRACT FROM AUTHOR]

Knowledge of RNA structure, either in isolation or in complex, is fundamental to understand the mechanism of cellular processes. Solid‐state NMR (ssNMR) is applicable to high molecular‐weight complexes and does not require crystallization; thus, it is well‐suited to study RNA as part of large multicomponent assemblies. Recently, we solved the first structures of both RNA and an RNA‐protein complex by ssNMR using conventional 13C‐ and 15N‐detection. This approach is limited by the severe overlap of the RNA peaks together with the low sensitivity of multidimensional experiments. Here, we overcome the limitations in sensitivity and resolution by using 1H‐detection at fast MAS rates. We develop experiments that allow the identification of complete nucleobase spin‐systems together with their site‐specific base pair pattern using sub‐milligram quantities of one uniformly labelled RNA sample. These experiments provide rapid access to RNA secondary structure by ssNMR in protein‐RNA complexes of any size. [ABSTRACT FROM AUTHOR]

Plant cell walls consist of a mixture of polysaccharides that render the cell wall a strong and dynamic material. Understanding the molecular structure and dynamics of wall polysaccharides is important for understanding and improving the properties of this energy-rich biomaterial. So far, solid-state NMR studies of cell wall structure and dynamics have solely relied on 13C chemical shifts measured from 2D and 3D correlation experiments. To increase the spectral resolution, sensitivity and upper limit of measurable distances, it is of interest to explore 1H chemical shifts and 1H-detected NMR experiments for analyzing cell walls. Here we demonstrate 2D and 3D 1H–13C correlation experiments at both moderate and fast MAS frequencies of 10–50 kHz to resolve and assign 1H chemical shifts of matrix polysaccharides in Arabidopsis primary cell walls. Both 13C-detected and 1H-detected experiments are implemented and are shown to provide useful and complementary information. Using the assigned 1H chemical shifts, we measured long-range correlations between matrix polysaccharides and cellulose using 1H–1H instead of 13C–13C spin diffusion, and the 2D experiments can be conducted with either 13C or 1H detection. [ABSTRACT FROM AUTHOR]

Highlights • CP-based experiments probe rigid regions of membrane proteins. • INEPT-base experiments image dynamic regions of membrane proteins. • Dual acquisition enables the simultaneous detection of rigid and dynamics regions. • Solid-state NMR detects ground and excited states of membrane proteins. Abstract The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions. Here, we review our recent efforts to develop single and dual acquisition pulse sequences with either 1H or 13C detection that utilize both DC and J-coupling mediated transfer to detect both rigid and mobile regions of membrane proteins in native-like lipid environments. We show the application of these new methods for studying the conformational equilibrium of a single-pass membrane protein, phospholamban, which regulates the calcium transport across the sarcoplasmic reticulum (SR) membrane by interacting with the SR Ca2+-ATPase. We anticipate that these methods will be ideal to portray the complex dynamics of membrane proteins in their native environments. [ABSTRACT FROM AUTHOR]

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on 13C- and 15N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and 1H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 102 to 103, thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, 1H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively 2H-labeled proteins. [ABSTRACT FROM AUTHOR]

Solid-state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane-bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, 1 H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance-specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi-acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D 1 H-detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de-excitation of specific resonances were achieved by high-fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U- 13 C, 15 N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel 1 H-detected multi-acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems., (© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, 1 H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13 C, 15 N-enriched, and fully protonated samples of M 0 Aβ 1-42 fibrils by high-field 1 H-detected NMR at 23.4 T and 18.8 T, and 13 C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M 0 Aβ 1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1 H detection. These results suggest that 1 H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

• 1H detection was applied in high resolution solid state NMR experiments with an oriented protein sample. • The application of a solvent saturation pulse during the z-filter sufficiently suppress water signal. • 1H detection provides two-fold sensitivity enhancement on the Pf1 phage coat protein sample. • Sensitivity enhancement is affected by 15N resonances and dipolar coupling frequency line-widths of the protein sample. Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples. [ABSTRACT FROM AUTHOR]

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.