Magnesium(II)-ATP Complexes in 1-Ethyl-3-Methylimidazolium Acetate Solutions Characterized by 31 Mg ß-Radiation-Detected NMR Spectroscopy.

The complexation of MgII with adenosine 5'-triphosphate (ATP) is omnipresent in biochemical energy conversion, but is difficult to interrogate directly. Here we use the spin- 1/2 ß-emitter 31 Mg to study MgII -ATP complexation in 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) solutions using ß-radiation-detected nuclear magnetic resonance (ß-NMR). We demonstrate that (nuclear) spin-polarized 31 Mg, following ion-implantation from an accelerator beamline into EMIM-Ac, binds to ATP within its radioactive lifetime before depolarizing. The evolution of the spectra with solute concentration indicates that the implanted 31 Mg initially bind to the solvent acetate anions, whereafter they undergo dynamic exchange and form either a mono- (31 Mg-ATP) or di-nuclear (31 MgMg-ATP) complex. The chemical shift of 31 Mg-ATP is observed up-field of 31 MgMg-ATP, in accord with quantum chemical calculations. These observations constitute a crucial advance towards using ß-NMR to probe chemistry and biochemistry in solution.

Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes.

Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

Elucidating the Molecular Interactions Occurring during Drug Precipitation of Weak Bases from Lipid-Based Formulations: A Case Study with Cinnarizine and a Long Chain Self-Nanoemulsifying Drug Delivery System.

The aim of this study was to investigate if molecular interactions between the weak base cinnarizine and lipolysis products were affecting the morphology of precipitated drug formed during in vitro lipolysis. In vitro lipolysis studies of a self-nanoemulsifying drug delivery system with or without cinnarizine were conducted. The digestion phases (aqueous phase and pellet phase) were separated by ultracentrifugation, and the pellet was isolated and lyophilized. The lyophilized pellets were examined by X-ray powder diffraction, (13)C solid-state nuclear magnetic resonance ((13)C NMR), (1)H liquid-state NMR ((1)H NMR) spectroscopy and differential scanning calorimetry (DSC). The (13)C NMR data indicated that the carbonyl groups and aliphatic part of the lipids undergo structural changes when the pellet contains cinnarizine. The (1)H NMR data suggests interactions occurring around the nitrogens on cinnarizine and the carboxylic group of fatty acids. DSC thermograms showed cinnarizine to be homogeneously incorporated into the lipids of the pellet, and no free amorphous cinnarizine was present. The three techniques (13)C NMR, (1)H NMR, and DSC complement each other and suggest interactions to occur between fatty acids and cinnarizine, which in turn favors amorphous precipitation.

Specificity of the Metalloregulator CueR for Monovalent Metal Ions: Possible Functional Role of a Coordinated Thiol?

Metal-ion-responsive transcriptional regulators within the MerR family effectively discriminate between mono- and divalent metal ions. Herein we address the origin of the specificity of the CueR protein for monovalent metal ions. Several spectroscopic techniques were employed to study Ag(I) , Zn(II) , and Hg(II) binding to model systems encompassing the metal-ion-binding loop of CueR from E. coli and V. cholerae. In the presence of Ag(I) , a conserved cysteine residue displays a pKa  value for deprotonation of the thiol that is close to the physiological pH value. This property is only observed with the monovalent metal ion. Quantum chemically optimized structures of the CueR metal site with Cys 112 protonated demonstrate that the conserved Ser 77 backbone carbonyl oxygen atom from the other monomer of the homodimer is "pulled" towards the metal site. A common allosteric mechanism of the metalloregulatory members of the MerR family is proposed. For CueR, the mechanism relies on the protonation of Cys 112.

Billion-fold enhancement in sensitivity of nuclear magnetic resonance spectroscopy for magnesium ions in solution.

ß-nuclear magnetic resonance (NMR) spectroscopy is highly sensitive compared to conventional NMR spectroscopy, and may be applied for several elements across the periodic table. ß-NMR has previously been successfully applied in the fields of nuclear and solid-state physics. In this work, ß-NMR is applied, for the first time, to record an NMR spectrum for a species in solution. (31)Mg ß-NMR spectra are measured for as few as 10(7) magnesium ions in ionic liquid (EMIM-Ac) within minutes, as a prototypical test case. Resonances are observed at 3882.9 and 3887.2 kHz in an external field of 0.3 T. The key achievement of the current work is to demonstrate that ß-NMR is applicable for the analysis of species in solution, and thus represents a novel spectroscopic technique for use in general chemistry and potentially in biochemistry.

Exploring the solid-form landscape of pharmaceutical hydrates: transformation pathways of the sodium naproxen anhydrate-hydrate system.

PURPOSE: To understand the transformation pathways amongst anhydrate/hydrate solid forms of sodium naproxen and to highlight the importance of a polymorphic dihydrate within this context. METHODS: Multi-temperature dynamic vapour sorption (DVS) analysis combined with variable-humidity X-ray powder diffraction (XRPD) to establish the transformation pathways as a function of temperature and humidity. XRPD and thermogravimetric analysis (TGA) to characterise bulk samples. Monitoring of in-situ dehydration using solid-state (13)C CP/MAS spectroscopy. RESULTS: At 25 °C, anhydrous sodium naproxen (AH) transforms directly to one dihydrate polymorph (DH-II). At 50 °C, AH transforms stepwise to a monohydrate (MH) then to the other dihydrate polymorph (DH-I). DH-II transforms to a tetrahydrate (TH) more readily than DH-I transforms to TH. Both dihydrate polymorphs transform to the same MH. CONCLUSIONS: The properties of the polymorphic dihydrate control the transformation pathways of sodium naproxen.

Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 µM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

Monitoring the stability of heparin: NMR evidence for the rearrangement of sulfated iduronate in phosphate buffer.

Heparin, a major anticoagulant drug, comprises a complex mixture of motifs. Heparin is isolated from natural sources while being subjected to a variety of conditions but the detailed effects of these on heparin structure have not been studied in depth. Therefore, the result of exposing heparin to a range of buffered environments, ranging pH values from 7 to 12, and temperatures of 40, 60 and 80 °C were examined. There was no evidence of significant N-desulfation or 6-O-desulfation in glucosamine residues, nor of chain scission, however, stereochemical re-arrangement of α-L-iduronate 2-O-sulfate to α-L-galacturonate residues occurred in 0.1 M phosphate buffer at pH 12/80 °C. The results confirm the relative stability of heparin in environments like those during extraction and purification processes; on the other hand, the sensitivity of heparin to pH 12 in buffered solution at high temperature is highlighted, providing an important insight for heparin manufacturers.

Residue specific hydration of primary cell wall potato pectin identified by solid-state 13C single-pulse MAS and CP/MAS NMR spectroscopy.

Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by (13)C single-pulse (SP) magic-angle-spinning (MAS) and (13)C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by (2)H SP/MAS NMR experiments. The study shows that the arabinan side chains hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose-galacturonic acid (Rha-GalA) backbone in RG-I. Potential food ingredient uses of RG-I by tailoring of its structure are discussed.

Exciting Opportunities for Solid-State 95Mo NMR Studies of MoS2 Nano-structures in Materials Research from Low to Ultra-high Magnetic Field (35.2 T).

Solid-state, natural-abundance 95Mo NMR experiments of four different MoS2 materials have been performed on a magnet B 0 = 19.6 T and on a new Series Connected Hybrid (SCH) magnet at 35.2 T. Employing two different 2H-MoS2 (2H phase) materials, a "pseudo-amorphous" MoS2 nano-material, and a MoS2 layer on the Al2O3 support of a hydrodesulphurization (HDS) catalyst have enabled introduction of solid-state 95Mo NMR as an important analytical tool in studies of MoS2 nano-materials. 95Mo spin-lattice relaxation time (T 1) studies of 160- and 4-layer 2H-MoS2 samples at 19.6 and 35.2 T show their relaxation rates (1/T 1) increase in proportion to B 0 2. This is in accord with chemical shift anisotropy (CSA) relaxation being the dominant T 1(95Mo) mechanism, with a large 95Mo CSA = 1025 ppm determined for all four MoS2 nano-materials. The dominant CSA mechanism suggests the MoS2 band-gap electrons are delocalized throughout the lattice-layer structures, thereby acting as a fast modulation source (ω oτc << 1) for 95Mo CSA in 2H-MoS2. A decrease in T 1(95Mo) is observed for an increase in B 0 field and for a decrease in the number of 2H-MoS2 layers. All four nano-materials exhibit identical 95Mo electric field gradient (EFG) parameters. The T 1 results account for the several failures to retrieve 95Mo spectral EFG and CSA parameters for multilayer 2H-MoS2 samples in the pioneering solid-state 95Mo NMR studies performed during the past two decades (1990-2010), because of the extremely long T 1(95Mo) = ~200-250 s observed at low B 0 (~9.4 T) used at that time. Much shorter T 1(95Mo) values are observed even at 19.6 T for the "pseudo-amorphous" and the HDS catalyst (MoS2-Al2O3 support) MoS2 nano-materials. These allowed useful solid-state 95Mo NMR spectra for these two samples to be obtained at 19.6 T in a few to < 24 h. Most importantly, this research led to observation of an impressive 95Mo MAS spectrum for an average of 1-4 thick MoS2-layers on a Al2O3 support, i.e., the first MAS NMR spectrum of a low natural-abundance, low-γ quadrupole-nucleus species layered on a catalyst support. While a huge gain in NMR sensitivity, factor ~ 60, is observed for the 95Mo MAS spectrum of the 160-layer sample at 35.2 T compared to 14.1 T, the MAS spectrum for the 4-layer sample is almost completely wiped out at 35.2 T. This unusual observation for the 4-layer sample (crumpled, rose-like and defective Mo-edge structures) is due to an increased distribution of the isotropic 95Mo shifts in the 95Mo MAS spectra at B 0 up to 35.2 T upon reduction of the number of sample layers.

Fast measurement of phosphates and ammonium in fermentation-like media: A feasibility study.

Real-time monitoring of bioprocesses plays a key-role in modern industries, providing new information on full-scale production, thus enabling control of the process and allowing it to run at optimal conditions while minimizing waste. Monitoring of phosphates and ammonium in fermentation processes has a twofold interest: they are important nutrients for living organisms while at the same time constituting environmental nutrient pollutants, for which unnecessary use and disposal must be avoided. In this report, the possibility of simultaneous analysis of phosphates and ammonium in fermentations was verified using spectroscopy-based methods combined with chemometrics to construct calibration models. To achieve this, the models were based on synthetic samples mimicking real fermentation media, providing a dataset where the analytes were completely uncorrelated. Different at-line techniques (mid- and near- infrared spectroscopy, MIR and NIR) were evaluated for their ability to monitor quickly both analytes, in a wide range of concentrations (10-100 mM), in three media of different complexities. Partial Least Squares (PLS) models on MIR spectroscopy gave very good results, with prediction errors lower than 5 % for both analytes in all datasets. In contrast, the results for PLS models on NIR spectroscopy were inferior (prediction errors between 3 and 26 %) for both analytes, as, in the case of phosphate, it could be demonstrated that the model was based on based on indirect predictions.

Quantitation of Protein Cysteine-Phenol Adducts in Minced Beef Containing 4-Methyl Catechol.

Thiol groups of cysteine (Cys) residues in proteins react with quinones, oxidation products of polyphenols, to form protein-polyphenol adducts. The aim of the present work was to quantify the amount of adduct formed between Cys residues and 4-methylcatechol (4MC) in minced beef. A Cys-4MC adduct standard was electrochemically synthesized and characterized by liquid chromatography-mass spectrometry (LC-MS) as well as NMR spectroscopy. Cys-4MC adducts were quantified after acidic hydrolysis of myofibrillar protein isolates (MPIs) and LC-MS/MS analysis of meat containing either 500 or 1500 ppm 4MC and stored at 4 °C for 7 days under a nitrogen or oxygen atmosphere. The concentrations of Cys-4MC were found to be 2.2 ± 0.3 nmol/mg MPI and 8.1 ± 0.9 nmol/mg MPI in meat containing 500 and 1500 ppm 4MC, respectively, and stored for 7 days under oxygen. The formation of the Cys-4MC adduct resulted in protein thiol loss, and ca. 62% of the thiol loss was estimated to account for the formation of the Cys-4MC adduct for meat containing 1500 ppm 4MC. Furthermore, protein polymerization increased in samples containing 4MC as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the polymerization was found to originate from protein-polyphenol interactions as evaluated by a blotting assay with staining by nitroblue tetrazolium.

Hg2+ and Cd2+ binding of a bioinspired hexapeptide with two cysteine units constructed as a minimalistic metal ion sensing fluorescent probe.

Hg2+ and Cd2+ complexation of a short hexapeptide, Ac-DCSSCY-NH2 (DY), was studied by pH-potentiometry, UV and NMR spectroscopy and fluorimetry in aqueous solutions and the Hg2+-binding ability of the ligand was also described in an immobilized form, where the peptides were anchored to a hydrophilic resin. Hg2+ was demonstrated to form a 1 : 1 complex with the ligand even at pH = 2.0 while Cd2+ coordination by the peptide takes place only above pH ∼ 3.5. Both metal ions form bis-ligand complexes by the coordination of four Cys-thiolates at ligand excess above pH ∼ 5.5 (Cd2+) and 7.0 (Hg2+). Fluorescence studies demonstrated a Hg2+ induced concentration-dependent quenching of the Tyr fluorescence until a 1 : 1 Hg2+ : DY ratio. The fluorescence emission intensity decreases linearly with the increasing Hg2+ concentration in a range of over two orders of magnitude. The fact that this occurs even in the presence of 1.0 eq. of Cd2+ per ligand reflects a complete displacement of the latter metal ion by Hg2+ from its peptide-bound form. The immobilized peptide was also shown to bind Hg2+ very efficiently even from samples at pH = 2.0. However, the existence of lower affinity binding sites was also demonstrated by binding of more than 1.0 eq. of Hg2+ per immobilized DY molecule under Hg2+-excess conditions. Experiments performed with a mixture of four metal ions, Hg2+, Cd2+, Zn2+ and Ni2+, indicate that this molecular probe may potentially be used in Hg2+-sensing systems under acidic conditions for the measurement of µM range concentrations.

1H NMR and LC-MS-based metabolomic approach for evaluation of the seasonality and viticultural practices in wines from São Francisco River Valley, a Brazilian semi-arid region.

São Francisco River Valley (SFRV) is a wine-producing semi-arid region in Brazil. Therefore, we used a 1H NMR and UPLC-MS-based metabolomic approach coupled to chemometrics to evaluate the variability in Chenin Blanc and Syrah wines for two harvest seasons, two vine training system and six rootstocks. Overall, the secondary metabolites were influenced by the three factors studied, whereas the primary metabolites were only by the seasonality. Chenin Blanc wines made in December presented higher content of an unidentified carbohydrate. In Syrah wines, glycerol, tartaric acid, succinic acid and 2,3-butanediol were greater in December, while proline and lactic acid were more abundant in July. For training system, caffeic acid derivatives were increased in wines produced from espalier. Lyre system increased phenolic compounds, organic acids and apocarotenoids. The effect of the rootstocks was less pronounced, affecting basically caffeic acid derivatives. Thus, we expect that our results may assist the winemakers to improve the SFRV wine quality.

Direct observation of Mg2+ complexes in ionic liquid solutions by 31Mg ß-NMR spectroscopy.

NMR spectra of Mg2+ ions in ionic liquids were recorded using a highly sensitive variant of NMR spectroscopy known as ß-NMR. The ß-NMR spectra of MgCl2 in EMIM-Ac and EMIM-DCA compare favourably with conventional NMR, and exhibit linewidths of ∼3 ppm, allowing for discrimination of species with oxygen and nitrogen coordination.

Order and disorder in titanosilicate glass by 17O MAS, off-MAS, and 3Q-QCPMG-MAS solid-state NMR.

An 17O-enriched version of the titanosilicate glass, KTS2 (K(2)O.TiO(2).2SiO(2)), was analyzed by 17O MAS, off-MAS, and 3Q-QCPMG-MAS experiments. Exploiting the variations in EFG and CSA parameters for the 17O sites in KTS2 glass, we detected four types of oxygen by reduction of spinning sideband intensities in the off-MAS experiments. From the 17O off-MAS and 3Q-QCPMG-MAS experiments, the Si-O-Ti and K-O-Ti resonances were characterized by a distribution of isotropic chemical shifts, whereas the Si-O-Si resonance was characterized by very small distributions of both EFG tensor and isotropic chemical shift, which means that the disorder in the glass is closely related to Ti. In addition to the order/disorder issue, the most striking feature about the 17O off-MAS experiments on KTS2 is the lack of signals from Ti-O-Ti, which contradicts linking between corner sharing TiO(5) units. Therefore, the structure must consist of linkages between TiO(5) units and SiO(4) tetrahedra and linkages between SiO(4) tetrahedra.

1H NMR spectra dataset and solid-state NMR data of cowpea (Vigna unguiculata).

In this article the NMR data from chemical shifts, coupling constants, and structures of all the characterized compounds were provided, beyond a complementary PCA evaluation for the corresponding manuscript (E.G. Alves Filho, L.M.A. Silva, E.M. Teofilo, F.H. Larsen, E.S. de Brito, 2017) [3]. In addition, a complementary assessment from solid-state NMR data was provided. For further chemometric analysis, numerical matrices from the raw 1H NMR data were made available in Microsoft Excel workbook format (.xls).

Solubilization of industrial grade plant protein by enzymatic hydrolysis monitored by vibrational and nuclear magnetic resonance spectroscopy: A feasibility study.

Protein hydrolysates are of great interest in the food industry due to their nutritional and functional properties, but their use often implies solubilization in water and therefore hamper the use of plant proteins with inherent low water solubility. Protein solubility in water can be modified by enzymatic hydrolysis, but during this process several collateral properties of the protein hydrolysates changes. It is therefore important to determine the end-point of the process and to monitor its development. In this feasibility study, we demonstrated the potential of different spectroscopic techniques (1H NMR and IR) coupled with chemometrics analysis in monitoring the hydrolysis of five different industrial grade plant proteins by the enzyme Alcalase. Logarithmic modeling of the PCA (Principal Component Analysis) scores confirmed that they can represent a measurement of the solubilized protein material released and resulted in kinetic parameters describing the suitability of protein sources as substrates for the hydrolysis. This way, we showed that a qualitative evaluation of the degree of hydrolysis is possible using fast at-line technologies and PCA.

Genotype evaluation of cowpea seeds (Vigna unguiculata) using 1H qNMR combined with exploratory tools and solid-state NMR.

The ultimate aim of this study was to apply a non-targeted chemometric analysis (principal component analysis and hierarchical clustering analysis using the heat map approach) of NMR data to investigate the variability of organic compounds in nine genotype cowpea seeds, without any complex pre-treatment. In general, both exploratory tools show that Tvu 233, CE-584, and Setentão genotypes presented higher amount mainly of raffinose and Tvu 382 presented the highest content of choline and least content of raffinose. The evaluation of the aromatic region showed the Setentão genotype with highest content of niacin/vitamin B3 whereas Tvu 382 with lowest amount. To investigate rigid and mobile components in the seeds cotyledon, 13C CP and SP/MAS solid-state NMR experiments were performed. The cotyledon of the cowpea comprised a rigid part consisting of starch as well as a soft portion made of starch, fatty acids, and protein. The variable contact time experiment suggests the presence of lipid-amylose complexes.

Separation of 47Ti and 49Ti solid-state NMR lineshapes by static QCPMG experiments at multiple fields.

Experimental procedures are proposed and demonstrated that separate the spectroscopic contribution from both (47)Ti and (49)Ti in solid-state nuclear magnetic resonance spectra. These take advantage of the different nuclear spin quantum numbers of these isotopes that lead to different "effective" radiofrequency fields for the central transition nutation frequencies when these nuclei occur in sites with a significant electric field gradient. Numerical simulations and solid-state NMR experiments were performed on the TiO(2) polymorphs anatase and rutile. For anatase, the separation of the two isotopes at high field (21.1T) facilitated accurate determination of the electric field gradient (EFG) and chemical shift anisotropy (CSA) tensors. This was accomplished by taking advantage of the quadrupolar interaction between the EFG at the titanium site and the different magnitudes of the nuclear quadrupole moments (Q) of the two isotopes. Rutile, having a larger quadrupolar coupling constant (C(Q)), was examined by (49)Ti-selective experiments at different magnetic fields to obtain spectra with different scalings of the two anisotropic tensors. A small chemical shielding anisotropy (CSA) of -30 ppm was determined.