The role of 1H solid-state NMR in structure elucidation of solids is becoming more preponderant, particularly as faster magic-angle spinning rates (MAS) become available which improve 1H detected assignment strategies. However, current 1H spectral resolution is still relatively poor, with linewidths of typically a few hundred Hz, even at the fastest rates available today. Here we detail and assess the factors limiting proton linewidths and line shapes in MAS experiments with five different samples, exemplifying the different sources of broadening that affect the residual linewidth. We disentangle the different contributions through one- and two-dimensional experiments: by using dilution to identify the contribution of ABMS; by using extensive deuteration to identify the dipolar contributions; and by using variable MAS rates to determine the ratio between homogeneous and inhomogeneous components. We find that the overall widths and the nature of the different contributions to the linewidths can vary very considerably. While we find that faster spinning always yields narrower lines and longer coherence lifetimes, we also find that for some resonances the dipolar contribution is no longer dominant at 100 kHz MAS. When the inhomogeneous sources of broadening, such as ABMS and chemical shift disorder, are dominant, two-dimensional 1H-1H correlation experiments yield better resolution for assignment. Particularly the extraction of the antidiagonal of a 2D peak will remove any correlated inhomogeneous broadening, giving substantially narrower 1H linewidths.

The advent of magic angle spinning (MAS) rates exceeding 100 kHz has facilitated the acquisition of 1H-detected solid-state NMR spectra of biomolecules with high resolution. However, challenges can arise when preparing rotors for these experiments, due to the physical properties of biomolecular solid samples and the small dimensions of the rotors. In this study, we have designed 3D-printable centrifugal devices that facilitate efficient and consistent packing of crystalline protein slurries or viscous phospholipids into 0.7 mm rotors. We demonstrate the efficacy of these packing devices using 1H-detected solid state NMR at 105 kHz. In addition to devices for 0.7 mm rotors, we have also developed devices for other frequently employed rotor sizes and styles. We have made all our designs openly accessible, and we encourage their usage and ongoing development as a shared effort within the solid state NMR community.

In the last three decades, the scope of solid-state NMR has expanded to exploring complex biomolecules, from large protein assemblies to intact cells at atomic-level resolution. This diversity in macromolecules frequently features highly flexible components whose insoluble environment precludes the use of solution NMR to study their structure and interactions. While High-resolution Magic-Angle Spinning (HR-MAS) probes offer the capacity for gradient-based 1H-detected spectroscopy in solids, such probes are not commonly used for routine MAS NMR experiments. As a result, most exploration of the flexible regime entails either 13C-detected experiments, the use of partially perdeuterated systems, or ultra-fast MAS. Here we explore proton-detected pulse schemes probing through-bond 13C-13C networks to study mobile protein sidechains as well as polysaccharides in a broadband manner. We demonstrate the use of such schemes to study a mixture of microtubule-associated protein (MAP) tau and human microtubules (MTs), and the cell wall of the fungus Schizophyllum commune using 2D and 3D spectroscopy, to show its viability for obtaining unambiguous correlations using standard fast-spinning MAS probes at high and ultra-high magnetic fields.

The resolution of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra remains bounded by the spinning frequency, which is limited by the material strength of MAS rotors. Since diamond is capable of withstanding 1.5-2.5x greater MAS frequencies, compared to state-of-the art zirconia, we fabricated rotors from single crystal diamond. When combined with bearings optimized for spinning with helium gas, diamond rotors could achieve the highest MAS frequencies to date. Furthermore, the excellent microwave transmission properties and thermal conductivity of diamond could improve sensitivity enhancements in dynamic nuclear polarization (DNP) experiments. The fabrication protocol we report involves novel laser micromachining and produced rotors that presently spin at ωr/2π = 111.000 ± 0.004 kHz, with stable spinning up to 124 kHz, using N2 gas as the driving fluid. We present the first proton-detected 13C/15N MAS spectra recorded using diamond rotors, a critical step towards studying currently inaccessible ex-vivo protein samples with MAS NMR. Previously, the high aspect ratio of MAS rotors (∼10:1) precluded fabrication of MAS rotors from diamond.

One key bottleneck of solid-state NMR spectroscopy is that 1 H NMR spectra of organic solids are often very broad due to the presence of a strong network of dipolar couplings. We have recently suggested a new approach to tackle this problem. More specifically, we parametrically mapped errors leading to residual dipolar broadening into a second dimension and removed them in a correlation experiment. In this way pure isotropic proton (PIP) spectra were obtained that contain only isotropic shifts and provide the highest 1 H NMR resolution available today in rigid solids. Here, using a deep-learning method, we extend the PIP approach to a second dimension, and for samples of L-tyrosine hydrochloride and ampicillin we obtain high resolution 1 H-1 H double-quantum/single-quantum dipolar correlation and spin-diffusion spectra with significantly higher resolution than the corresponding spectra at 100 kHz MAS, allowing the identification of previously overlapped isotropic correlation peaks.

The effects of two cationic peptides on phospholipid lateral diffusion in binary mixtures of POPC with various anionic phospholipids were measured via 31P CODEX NMR. Large unilamellar vesicles composed of POPC/POPG (70/30 mol/mol), or POPC/DOPS (70/30 mol/mol), or POPC/TOCL (85/15 mol/mol), or POPC/DOPA (50/50 mol/mol) were exposed to either polylysine (pLYS, N = 134 monomers) or KL-14 (KKLL KKAKK LLKKL), a model amphipathic helical peptide, in an amount corresponding to 80% neutralization of the anionic phospholipid charge by the cationic lysine residues. In the absence of added peptide, phospholipid lateral diffusion coefficients (all measured at 10 °C) increased with increasing reduced temperature (T-Tm). The POPC/DOPA mixture was an exception to this generalization, in that lateral diffusion for both components was far slower than any other mixture investigated, an effect attributed to intermolecular hydrogen bonding. The addition of pLYS or KL-14 decreased lateral diffusion in the POPC/DOPS LUV, but had minimal effects in the POPC/POPG LUV, indicating that ease of access of the cationic peptide residues to the anionic phospholipid groups was important. Both cationic peptides produced the opposite effect in the POPC/DOPA case, in that lateral diffusion increased significantly in their presence, with KL-14 being most effective. This latter observation was interpreted in terms of the electrostatic / H-bond model proposed by Kooijman et al. [Journal of Biological Chemistry, 282:11356-11,364, 2007] to describe the mechanism of interaction between the phosphomonoester head group of PA and the tertiary amine of lysine.

Spherical rotors in magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments have potential advantages relative to cylindrical rotors in terms of ease of fabrication, low risk of rotor crash, easy sample exchange, and better microwave access. However, one major disadvantage so far of spherical rotors is poor NMR filling factor due to the small sample volume and large cylindrical radiofrequency (RF) coil. Here we present a novel NMR coil geometry in the form of a spherical coil. The spherical coil best fits the spherical sample to maximize sensitivity, while also providing excellent RF homogeneity. We further improve NMR sensitivity by employing a spherical shell as the rotor, thereby maximizing sample volume (219 μL in this case of 9.5 mm outer diameter spheres). The spinning gas is supplied by a 3D-printed ring stator external to the coil, thereby introducing a simplified form of MAS stators. In this apparatus, the RF field generated along the coil axis is perpendicular to the external magnetic field, regardless of rotor orientation. We observe a linear increase in sensitivity with increasing sample volume. We also simulate the RF performance of spherical and cylindrical solenoid coils with constant or variable pitch for spherical and cylindrical rotors, respectively. The simulation results show that spherical solenoid coils generate comparable B1 field intensities but have better homogeneity than cylindrical solenoid coils do.

Chromatin is a DNA-protein polymer that represents the functional form of the genome. The main building block of chromatin is the nucleosome, a structure that contains 147 base pairs of DNA and two copies each of the histone proteins H2A, H2B, H3 and H4. Previous work has shown that magic angle spinning (MAS) NMR spectroscopy can capture the nucleosome at high resolution although studies have been challenging due to low sensitivity, the presence of dynamic and rigid components, and the complex interaction networks of nucleosomes within the chromatin polymer. Here, we use dynamic nuclear polarization (DNP) to enhance the sensitivity of MAS NMR experiments of nucleosome arrays at 100 K and show that well-resolved 13C-13C MAS NMR correlations can be obtained much more efficiently. We evaluate the effect of temperature on the chemical shifts and linewidths in the spectra and demonstrate that changes are relatively minimal and clustered in regions of histone-DNA or histone-histone contacts. We also compare samples prepared with and without DNA and show that the low temperature 13C-13C correlations exhibit sufficient resolution to detect chemical shift changes and line broadening for residues that form the DNA-histone interface. On the other hand, we show that the measurement of DNP-enhanced 15N-13C histone-histone interactions within the nucleosome core is complicated by the natural 13C abundance network in the sample. Nevertheless, the enhanced sensitivity afforded by DNP can be used to detect long-range correlations between histone residues and DNA. Overall, our experiments demonstrate that DNP-enhanced MAS NMR spectroscopy of chromatin samples yields spectra with high resolution and sensitivity and can be used to capture functionally relevant protein-DNA interactions that have implications for gene regulation and genome organization.

G protein-coupled receptors (GPCRs) have a simple seven transmembrane helix architecture which has evolved to recognize a diverse number of chemical signals. The more than 800 GPCRs encoded in the human genome function as receptors for vision, smell and taste, and mediate key physiological processes. Consequently, these receptors are a major target for pharmaceuticals. Protein crystallography and electron cryo-microscopy have provided high resolution structures of many GPCRs in both active and inactive conformations. However, these structures have not sparked a surge in rational drug design, in part because GPCRs are inherently dynamic and the structural changes induced by ligand or drug binding to stabilize inactive or active conformations are often subtle rearrangements in packing or hydrogen-bonding interactions. NMR spectroscopy provides a sensitive probe of local structure and dynamics at specific sites within these receptors as well as global changes in receptor structure and dynamics. These methods can also capture intermediate states and conformations with low populations that provide insights into the activation pathways. We review the use of solid-state magic angle spinning NMR to address the structure and activation mechanisms of GPCRs. The focus is on the large and diverse class A family of receptors. We highlight three specific class A GPCRs in order to illustrate how solid-state, as well as solution-state, NMR spectroscopy can answer questions in the field involving how different GPCR classes and subfamilies are activated by their associated ligands, and how small molecule drugs can modulate GPCR activation.

3D printing has evolved into an invaluable tool for rapid and cost-effective production of intricate parts. In this paper we describe 3D printing and other rapid prototyping methods to fabricate 3.2 mm stators and drive caps for use in magic angle spinning (MAS) NMR experiments. These components can be fabricated with the assistance of computer-aided design (CAD) software and at a fraction of the cost of commercial parts. Additionally, we show that the performance of these 3D printed stators and drive caps is comparable to commercially available systems and that they have significant advantages over their machined counterparts.