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.

Glasses of enlarged chemical composition in the xCsBr-(100-x)B2O3 system, with x= 30-80 mol% have been prepared and studied for the first time. The hydrothermal preparation method is used to prepare fine powdered samples. The X- ray Diffraction (XRD), Fourier Transform Infrared spectra (FTIR) and 11B Nuclear Magnetic Resonance (11B NMR) are the tools used to study the structure of glasses. Even at the high CsBr concentration (30-60 mol%), XRD results revealed an amorphous structure characterized most of the examined compositions. But glasses enriched with CsBr (70 and 80 mol%) possess some ordered sub-structures impeded in the main amorphous network. 11B NMR and FTIR spectroscopy confirmed the presence of BO3 triangle as the major and dominant structural units. The four coordinated boron (BO4), on the other hand, can't be detected in the all analyzed compositions. An extremely low concentration from BBr4 tetrahedral groups (boron coupled with Br) is formed. The B-O bonds of planar BO3 triangles, which are the fundamental units, were involved in the production of amorphous clusters of the type Cs4B5O9]Br. Density (D) measurement and calculating molar volume (Vm), packing density and free volume(Vf) have been studied. The added CsBr can't play a role of modifier but it might be inserted interstitially between the borate structural units causing increasing of molar volume and free space in the glass network structure. The results based on transmission electron microscopy (TEM-EDP) and X-ray diffraction pattern (XRD) are in good agreement, indicating that the amorphous-clustered species are developed in the studied glasses.

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.

Icosahedral dsDNA viruses such as the tailed bacteriophages and herpesviruses have a conserved pathway to virion assembly that is initiated from a scaffolding protein driven procapsid formation. The dsDNA is actively packaged into procapsids, which undergo complex maturation reactions to form infectious virions. In bacteriophage P22, scaffolding protein (SP) directs the assembly of coat proteins into procapsids that have a T=7 icosahedral arrangement, en route to the formation of the mature P22 capsid. Other than the C-terminal helix-turn-helix involved in interaction with coat protein, the structure of the P22 303 amino acid scaffolding protein within the procapsid is not understood. Here, we present a structural model of P22 scaffolding protein encapsulated within the 23 MDa procapsid determined by magic angle spinning NMR spectroscopy. We took advantage of the 10-fold sensitivity gains afforded by the novel CPMAS CryoProbe to establish the secondary structure of P22 scaffolding protein and employed 19F MAS NMR experiments to probe its oligomeric state in the procapsid. Our results indicate that the scaffolding protein has both α-helical and disordered segments and forms a trimer of dimers when bound to the procapsid lattice. This work provides the first structural information for P22 SP beyond the C-terminal helix-turn-helix and demonstrates the power of MAS NMR to understand higher-order viral protein assemblies involving structural components that are inaccessible to other structural biology techniques.

This article presents a study of the attenuation of gamma rays in glassy materials and an Electron Paramagnetic Resonance (EPR) study of xAl2O3·(40−x)Ag2O·60P2O5 system where x varies between 0–20 mol%. The EPR study examined the effect of aluminum oxide on the EPR parameters of silver ions in phosphate glasses. The EPR spectra show that after gamma irradiation, additional signals are connected to silver-related species such as Ag0, Ag+, or Ag2+. Gamma rays with energies ranging from 118.27 to 1418.15 keV were used to investigate the attenuation coefficients in current glass samples with thicknesses up to 3 cm. The attenuation of gamma rays was investigated by measuring the mass attenuation coefficient (MAC), half-value layer (HVL), effective atomic number (Zeff), and exposure build-up factor of various glass samples with different aluminum oxide concentrations. The results showed that the MAC values for glasses with a high concentration of aluminum oxide were larger than those for binary glasses at high energies, indicating an improvement in shielding capacity. Also, the glasses studied in this work provide shorter HVL than RS-253 G18 commercial glasses and various concrete samples, including ordinary, Basalt-magnetite, hematite-serpentine, and ilmenite-limonite. These findings provide important insights into using phosphate glasses as gamma-ray shields and the effect of aluminum oxide on the properties of phosphate glasses containing silver ions.

67 Zn solid-state NMR suffers from low sensitivity, limiting its ability to probe the Zn2+ surroundings in MOFs. We report a breakthrough in overcoming challenges in 67Zn NMR. Combining new cryogenic MAS probe technology and performing NMR experiments at a high magnetic field results in remarkable signal enhancement, yielding enhanced information for MOF characterization.

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.

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.

<p>PDF file - 118K, Supplementary Figure S1. Species specificity of p14ARF and p19ARF antibodies. Supplementary Figure S2. Increase in SAβGal-positive cell numbers in control mice during early aging. Supplementary Figure S3. Rb and p130 dephosphorylation upon p14ARF induction. Supplementary Figure S4. Effects of p14ARF induction on skin histology. Supplementary Figure S5. p14ARF induces hair-follicle stem cell dysfunction through p53. Supplementary Figure S6. Senescence in TPA-treated mice.</p>

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.

We report the first “total synthesis” of 17O-labeled d-glucose and its solid-state 17O NMR characterization with unprecedented sensitivity and resolution.

Glass structure comprising (0-20) mol% Al2O3-(20-40) mol % Ag2O-and 60 mol % P2O5 was examined using a Al-27, P-31 MAS- NMR and FTIR spectroscopy. The structural changes within the complicated ternary phosphate glasses were correlated with that of a simple binary silver phosphate. Results show that aluminum acts primarily as intermediate ions, while silver acts as a potent modifier. The number of non-bridging oxygen atoms (NBO) on average in the structure of silver aluminophosphate (SAP) glasses decreases with increasing Al2O3 content. The aluminum ions in the SAP glass system occur in the coordinations of both Al (6) and Al (4). The Al (6) fraction rises with rising concentration of Al2O3. Al (6) is more than Al (4) in the glass containing more Al2O3 than that of 20 mol of Al2O3. The glass was transformed into a glass-ceramics by the effect of thermal heat treatment at 600 degrees C for 4 h.

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.

Timed artificial insemination (TAI) is a very important tool for reproductive programs in small ruminants, providing synchronized inseminations and more efficient use of superior males. The objectives of this study were to investigate the viability of different breeding protocols under sub-optimal conditions and out of the breeding season, and to compare the outcome of TAI in sheep with two different semen extenders. Ewes (n=575) were subjected to estrous synchronization by intravaginal sponges containing 30 mg of medroxyprogesterone acetate and PMSG. Ewes were divided into 5 groups according to the semen extender employed and to the breeding protocol: 1) egg yolk citrate AI reinforced by ram (EYC-RR); 2) Andromed AI reinforced by ram (AND-RR), where rams joined the flocks 3 hours after AI; 3) egg yolk citrate AI without ram exposition (EYC); 4) Andromed AI without ram exposition (AND). In all these groups vaginal AIs were performed 56±2 hours from sponge removal and PMSG injection; 5) natural mating (RAM) where rams were introduced 48 hours following sponge removal and PMSG injection. Pregnancy rate, parturition rate, and prolificacy were significantly higher (p<0.05) in the EYC-RR and AND-RR groups than in the EYC and AND groups. The lambing rate was not different between groups. No statistical differences were observed between breeding farms or inseminating rams. This study showed that TAI with fresh semen diluted either with egg yolk citrate or with AndroMed could be an effective and feasible tool to increase lamb production and improve genetic merits of the sheep population out of the breeding season.

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.

Solid-state nuclear magnetic resonance is a promising technique to probe bone mineralization and interaction of collagen protein in the native state. However, many of the developments are hampered due to the low sensitivity of the technique. In this article, we report solid-state nuclear magnetic resonance (NMR) experiments using the newly developed BioSolids CryoProbe™ to access its applicability for elucidating the atomic-level structural details of collagen protein in native state inside the bone. We report here approximately a fourfold sensitivity enhancement in the natural abundance 13 C spectrum compared with the room temperature conventional solid-state NMR probe. With the advantage of sensitivity enhancement, we have been able to perform natural abundance 15 N cross-polarization magic angle spinning (CPMAS) and two-dimensional (2D) 1 H-13 C heteronuclear correlation (HETCOR) experiments of native collagen within a reasonable timeframe. Due to high sensitivity, 2D 1 H/13 C HETCOR experiments have helped in detecting several short and long-range interactions of native collagen assembly, thus significantly expanding the scope of the method to such challenging biomaterials.

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 set of glass samples of nominal composition xFe2O3.(43-x)B2O3.25SiO2.30Na2O. 2Al2O3, (x ≤ 40 mol%) was prepared via an ordinary melt quenching route. The variation of structural and electrical characteristics of the prepared samples have been studied. Electrical conduction in glasses that contain Fe2O3 (≤ 20 mol%) is considered to be ionic. Otherwise, the polaronic conduction is dominant in glasses of higher Fe2O3 concentrations (>20 mol%). Energy dispersive x-ray analysis of glass containing 5 mol% Fe2O3 showed that only one type of modifying iron ions Fe3+ present in their octahedral coordination. Besides, glasses containing higher Fe2O3 content reveal the presence of a mixture of both ferrous and ferric ions (Fe2+ & Fe3+). A fraction of both tetrahedral (BO4 and FeO4) units and BO3 groups are determined from Fourier transform infra-red spectroscopic analysis (FTIR). Increasing the fraction of tetrahedral units and decreasing BO3 are considered as the main reasons in conductivity enhancements.