Solid-state NMR spectroscopy is increasingly used to investigate the structure and dynamics of a wide range of chemical, material, and biological systems. Although limited sensitivity has long posed a major challenge, the recently developed MAS cryoprobe substantially alleviates this limitation. By enhancing the signal-to-noise (S/N) ratio without requiring sample freezing, the MAS cryoprobe is particularly well suited for studies of non-isotropic systems, including rigid solids (e.g., amyloid fibrils), semi-solids (e.g., membrane mimetics), and soft materials (e.g., nanodiscs and hydrogels). In this study, we demonstrate the enhanced sensitivity of solid-state NMR experiments on α-synuclein fibrils using a MAS cryoprobe. Compared with a conventional MAS probe, substantial improvements in S/N were observed in CPMAS, refocused INEPT, and 2D 13C13C chemical-shift correlation spectra. The increased sensitivity enables the detection of slowly decaying signals in the indirect dimension, thereby accelerating the acquisition of high-resolution multidimensional solid-state NMR data. These results highlight the potential of MAS cryoprobes for structural studies of samples that are scarce, unstable, or transient, such as amyloid intermediaries.

Abstract Understanding enzyme function requires characterizing not only static structure but also dynamics and ligand interactions. NMR spectroscopy provides this insight at atomic resolution, yet for large proteins the difficulty of resonance assignment has largely confined such studies to systems below ∼ 50 kDa, or to observing only methyl groups. Here we present an integrated magic-angle spinning (MAS) and solution NMR study of the 134 kDa tetrameric malate dehydrogenase from Ignicoccus islandicus ( Ii MDH), an enzyme of particular interest as an evolutionary intermediate between allosteric lactate dehydrogenases and non-allosteric malate dehydrogenases. By combining high-dimensional (up to 4D) MAS NMR experiments on sedimented protein with solution NMR, we achieved 92% backbone heavy-atom assignment and 91% assignment of all Ile-δ 1 , Leu-δ 1 /-δ 2 , Val-γ 1 /-γ 2 , Met-ɛ and Thr-γ methyl groups. Building on these assignments, MAS NMR 15 N rotating-frame relaxation ( R 1 p ) measurements revealed pronounced microsecond-timescale backbone dynamics in functionally critical regions, including the catalytic loop and the mobile surface loop. Complementary methyl-axis order parameters from solution NMR identified additional flexible sites in the hydrophobic core. Chemical shift perturbation experiments upon addition of the substrate analogue oxamate, monitored via backbone 1 H- 15 N TROSY, revealed both active-site contacts and rearrangements of helices a2F and a3G, regions implicated in allosteric signal transmission. The integrated approach demonstrated here exploits the distinct strengths of MAS and solution NMR, and provides a comprehensive view of structure, dynamics, and substrate interactions in a large oligomeric enzyme that would not be accessible by either technique alone.

Coronavirus envelope (E) proteins form drug-targeted ion channels that cause virulence to infected cells. The Middle East respiratory syndrome (MERS) virus has high mortality rates, but its E structure and function are unknown. We report the single-channel conductance and structure of membrane-bound MERS E protein. MERS E conducts K+ ions with a unitary conductance of 113 picosiemens, fivefold larger than the conductance of severe acute respiratory syndrome coronavirus 2 E. Solid-state nuclear magnetic resonance data indicate that the MERS E transmembrane domain forms a five-helix bundle that spans the lipid bilayer. The amino-terminal helical interface features multiple interacting phenylalanine (Phe) residues and an asparagine (Asn), whereas the carboxyl-terminal channel pore contains Phe33. Mutation of Phe17 abolished K+ conductance, whereas mutations of Phe33 and Asn15 suppressed most channel activity. These results indicate that MERS E contains two Phe-centered ion-conduction apparatuses, which likely permeate ions through cation-π interactions, providing the structural basis for developing antiviral drugs to inhibit this pathogenic viroporin.

Solid-state NMR spectroscopy is increasingly applied to structural and dynamics studies across a broad range of chemical, material, and biological systems. Although sensitivity has traditionally been a major limitation, the recently developed MAS cryoprobe has been shown to substantially overcome this challenge. Its ability to enhance the signal-to-noise (S/N) ratio without requiring sample freezing makes it particularly attractive for investigating non-isotropic systems, including soft materials (e.g., hydrogels), semi-solids (e.g., membrane mimetics) and rigid solids (e.g., amyloid fibrils). In this study, we report on the enhanced sensitivity of solid-state NMR experiments on α-synuclein fibrils using a MAS cryoprobe. Nearly an order-of-magnitude improvement in S/N was observed in CPMAS, refocused-INEPT and 2D 13C-13C chemical shift correlation spectra of α-synuclein fibrils compared with data collected on a conventional MAS probe. The improved S/N enables the acquisition of slowly decaying signals in the indirect dimension, facilitating faster, high-resolution multidimensional solid-state NMR spectroscopy. We therefore anticipate that MAS cryoprobe will become increasingly valuable for structural studies a wide range of samples that are less abundant, less stable, or transient, such as amyloid intermediates.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.

Solid-state NMR has great potential for investigating molecular structure, dynamics, and organization of the stratum corneum, the outer 10-20 μm of the skin, but is hampered by the unfeasibility of isotope labelling as generally required to reach sufficient signal-to-noise ratio for the more informative multidimensional NMR techniques. In this preliminary study of pig stratum corneum at 35 °C and water-free conditions, we demonstrate that cryogenic probe technology offers sufficient signal boost to observe previously undetectable minor resonances that can be uniquely assigned to fluid cholesterol, ceramides, and triacylglycerols, as well as enables 1H-1H spin diffusion monitored by 2D 1H-13C HETCOR to estimate 1-100 nm distances between specific atomic sites on proteins and lipids. The new capabilities open up for future multidimensional solid-state NMR studies to answer long-standing questions about partitioning of additives, such as pharmaceutically active substances, between solid and liquid domains within the protein and lipid phases in the stratum corneum and the lipids of the sebum.

Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well as highly flexible parts. Magic-angle spinning NMR is a powerful method to obtain atomic-level information about intact cell walls. Here we investigate sensitivity and information content of different homonuclear 13 C- 13 C and heteronuclear 1 H- 15 N, 1 H- 13 C and 15 N- 13 C correlation experiments. We demonstrate that a CPMAS CryoProbe yields ca. 8-fold increased signal-to-noise over a room-temperature probe, or a ca. 3-4-fold larger per-mass sensitivity. The increased sensitivity allowed to obtain high-resolution spectra even on intact bacteria. Moreover, we compare resolution and sensitivity of 1 H MAS experiments obtained at 100 kHz vs. 55 kHz. Our study provides useful hints for choosing experiments to extract atomic-level details on cell-wall samples.

NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14ARF). Oncogenic stress induces p14ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14ARF by NPM1 are unclear because the structural features of the p14ARF-NPM1 complex remain elusive. Here we show that NPM1 sequesters p14ARF within phase-separated condensates, facilitating the assembly of p14ARF into a gel-like meso-scale network. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal domain of p14ARF. Those hydrophobic interactions promote phase separation with NPM1, enhance nucleolar partitioning of p14ARF, restrict p14ARF and NPM1 diffusion within condensates and in nucleoli, and reduce cell viability. Our structural model provides novel insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

Solid-state nuclear magnetic resonance (ssNMR) is a high-resolution and versatile spectroscopic tool for characterizing pharmaceutical solids. However, the inherent low sensitivity of NMR remains a significant challenge in the analysis of natural abundance drug substances and products. Here, we report, for the first time, the application of a CPMAS CryoProbe™ to improve the sensitivity of 13C and 15N detection by approximately 5 to 6 times for solid-state analysis of a commercial pharmaceutical drug posaconazole (POSA). The sensitivity enhancement enables two-dimensional (2D) 13C-13C and 1H-15N correlation experiments, which are otherwise time-prohibitive using regular MAS probes, for resonance assignment and structural elucidation. These polarization transfer and correlation experiments reveal drug-drug and drug-polymer interactions in amorphous POSA and its amorphous solid dispersion formulation. Our results demonstrated that the CPMAS CryoProbe™ can be widely applied for routine pharmaceutical analysis and advanced structural investigations with significantly enhanced efficiency and throughput.

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

Although titanosilicalite-1 (TS-1) is among the most successful oxidation catalysts used in industry, its active site structure is still debated. Recent efforts have mostly focused on understanding the role of defect sites and extraframework Ti. Here, we report the 47/49Ti signature of TS-1 and molecular analogues [Ti(OTBOS)4] and [Ti(OTBOS)3(OiPr)] using novel MAS CryoProbe to enhance the sensitivity. While the dehydrated TS-1 displays chemical shifts similar to those of molecular homologues, confirming the tetrahedral environment of Ti consistent with X-ray absorption spectroscopy, it is associated with a distribution of larger quadrupolar coupling constants, indicating an asymmetric environment. Detailed computational studies on cluster models highlights the high sensitivity of the NMR signatures (chemical shift and quadrupolar coupling constant) to small local structural changes. These calculations show that, while it will be difficult to distinguish mono- vs dinuclear sites, the sensitivity of the 47/49Ti NMR signature should enable distinguishing the Ti location among specific T site positions.

the ARF tumor suppressor (Alternative Reading Frame, p19Arf in mouse, p14Arf in human), is an arginine (Arg)-rich intrinsically disordered protein. During interphase, ARF is expressed at low levels and is localized to the granular component of the nucleolus through interactions with NPM1. NPM1-ARF complexes are required for the maintenance of ribosome biogenesis homeostasis. p14Arf and NPM1 associate with the nucleolar 60S preribosomal particle. ARF deletion results in a NPM1-dependent surge in ribosome biogenesis, protein synthesis and increased nucleolar size, three phenotypic hallmarks of cancer cells.

SignificanceThe determination of active site protonation states is critical for a full mechanistic understanding of enzymatic transformations. However, hydrogen atom positions are challenging to extract using the standard tools of structural biology. Here, we make use of a joint solid-state NMR, X-ray crystallography, and first-principles computational approach that enables the investigation of enzyme catalysis at this fine level of chemical detail. For tryptophan synthase, this allows us to peer along the reaction coordinates into and out of the α-aminoacrylate intermediate. Through this process, we are developing a high-resolution probe for structural biology that is keenly sensitive to hydrogen atom positions—complementing diffraction methods yet able to be applied under conditions of active catalysis in microcrystalline and non-crystalline materials.

Across the evolutionary history of insects, the shift from nitrogen-rich carnivore/omnivore diets to nitrogen-poor herbivorous diets was made possible through symbiosis with microbes. The herbivorous turtle ants Cephalotes possess a conserved gut microbiome which enriches the nutrient composition by recycling nitrogen-rich metabolic waste to increase the production of amino acids. This enrichment is assumed to benefit the host, but we do not know to what extent. To gain insights into nitrogen assimilation in the ant cuticle we use gut bacterial manipulation, 15N isotopic enrichment, isotope-ratio mass spectrometry, and 15N nuclear magnetic resonance spectroscopy to demonstrate that gut bacteria contribute to the formation of proteins, catecholamine cross-linkers, and chitin in the cuticle. This study identifies the cuticular components which are nitrogen-enriched by gut bacteria, highlighting the role of symbionts in insect evolution, and provides a framework for understanding the nitrogen flow from nutrients through bacteria into the insect cuticle.

Nucleophosmin (NPM1) is an abundant nucleolar protein that aids in the maturation of pre-ribosomal particles and participates in oncogenic stress responses through its interaction with the Alternative Reading Frame tumor suppressor (ARF). NPM1 mediates multiple mechanisms of phase separation which contribute to the liquid-like properties of nucleoli. And in response to oncogenic stress, ARF sequesters NPM1 in the nucleolus resulting in cell cycle arrest or apoptosis. However, the effects of ARF on the structure and dynamics of NPM1 are poorly understood. Here we show that NPM1 undergoes phase separation with p14ARF in vitro, forming condensates with viscoelastic material properties. We probed the structure and dynamics of NPM1 within the condensed phase using solution- and solid-state NMR spectroscopy. Our results demonstrate that within the condensed phase, the NPM1 oligomerization domain forms an immobile scaffold, while the central intrinsically disordered region retains structural disorder, albeit with a marked decrease in dynamics. These observations provide insights into the molecular basis of ARF's nucleolar tumor suppressor function.

Despite breakthroughs in MAS NMR hardware and experimental methodologies, sensitivity remains a major challenge for large and complex biological systems. Here, we report that 3-4 fold higher sensitivities can be obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe (TM)). Such intensity enhancements permit recording 2D and 3D experiments that are otherwise time-prohibitive, such as 2D N-15-N-15 proton-driven spin diffusion and N-15-C-13 double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of applications, especially for biological systems suffering from low intrinsic sensitivity and at physiological temperatures. (C) 2019 Elsevier Inc. All rights reserved.

Nucleophosmin (NPM1) is an abundant nucleolar protein that aids in the maturation of pre-ribosomal particles and participates in oncogenic stress responses through its interaction with the Alternative Reading Frame tumor suppressor (p14ARF). NPM1 mediates multiple mechanisms of phase separation which contribute to the liquid-like properties of nucleoli. However, the effects of phase separation on the structure and dynamics of NPM1 are poorly understood. Here we show that NPM1 undergoes phase separation with p14ARF in vitro, forming condensates that immobilize both proteins. We probed the structure and dynamics of NPM1 within the condensed phase using solid-state NMR spectroscopy. Our results demonstrate that within the condensed phase, the NPM1 oligomerization domain forms an immobile scaffold, while the central intrinsically disordered region and the C-terminal nucleic acid binding domain exhibit relative mobility.

A Correction to this paper has been published: https://doi.org/10.1038/s42004-020-00436-4

We propose a dipolar HMQC Hadamard-encoded (D-HMQC-Hn) experiment for fast 2D correlations of abundant nuclei in solids. The main limitation of the Hadamard methods resides in the length of the encoding pulses, which results from a compromise between the selectivity and the sensitivity due to losses. For this reason, these methods should mainly be used with sparse spectra, and they profit from the increased separation of the resonances at high magnetic fields. In the case of the D-HMQC-Hn experiments, we give a simple rule that allows directly setting the optimum length of the selective pulses, versus the minimum separation of the resonances in the indirect dimension. The demonstration has been performed on a fully 13C,15N labelled f-MLF sample, and it allowed recording the build-up curves of the 13C-15N cross-peaks within 10 min. However, the method could also be used in the case of less sensitive samples, but with more accumulations.