High-resolution NMR Spectroscopy Research Articles

NMR Reveals Light-Induced Changes in the Dynamics of a Photoswitchable Fluorescent Protein

The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins and their correlation with the observed photochemical and photophysical properties.

Highly reactive nanomineral assembly in soil colloids: Implications for paddy soil carbon storage

Mineral availability for carbon (C) binding is a key regulator of soil C storage, yet little is known about the highly reactive nanomineral assembly in the paddy soil colloids. Here, using high-resolution transmission electron microscopy (HRTEM), solid-state 27Al and 29Si nuclear magnetic resonance (NMR) spectroscopy and X-ray photoelectron spectroscopy (XPS), we investigated the coordination nature of short-range-ordered (SRO) minerals in water-dispersible colloids that were isolated from the paddy soil under different six-year fertilization regimes. Our results showed that organic fertilization not only promoted the transformation of crystalline minerals to SRO phases in the bulk soils but also increased the concentrations of Fe, Al and Si in the soil colloids compared to chemical fertilization alone, and thus enhanced the accumulation of organic C in both the bulk soils and the soil colloids. The HRTEM images revealed that water-dispersible colloids in all soils, regardless of treatment, were composed of crystalline Fe nanominerals (with some Al/Si) and SRO-Al/Si nanominerals (with some Fe) associated with organic C. Furthermore, the combined results from the 27Al and 29Si NMR spectroscopy and XPS not only confirmed the presence of SRO-Al/Si nanoparticles as Si-rich allophane and phytolith but also demonstrated that organic fertilization promoted the transformation of aluminosilicates to SRO-Al/Si nanominerals in soil colloids. Together, these findings indicate that six-year organic fertilization promotes the formation of SRO minerals (e.g., ferrihydrite, Si-rich allophane and Fe-substituted allophane, as well as phytolith) in soils and modulates the assembly of organo-mineral complexes possibly by driving the biogeochemical cycles of Fe, Al, Si and specific organic ligands, thus contributing to the long-term storage of C in paddy soils.

Spot and Cumulative Urine Samples Are Suitable Replacements for 24-Hour Urine Collections for Objective Measures of Dietary Exposure in Adults Using Metabolite Biomarkers

Measurement of multiple food intake exposure biomarkers in urine may offer an objective method for monitoring diet. The potential of spot and cumulative urine samples that have reduced burden on participants as replacements for 24-h urine collections has not been evaluated. The aim of this study was to determine the utility of spot and cumulative urine samples for classifying the metabolic profiles of people according to dietary intake when compared with 24-h urine collections in a controlled dietary intervention study. Nineteen healthy individuals (10 male, 9 female, aged 21-65 y, BMI 20-35 kg/m2) each consumed 4 distinctly different diets, each for 1 wk. Spot urine samples were collected ∼2 h post meals on 3 intervention days/wk. Cumulative urine samples were collected daily over 3 separate temporal periods. A 24-h urine collection was created by combining the 3 cumulative urine samples. Urine samples were analyzed with metabolite fingerprinting by both high-resolution flow infusion electrospray mass spectrometry (FIE-HRMS) and proton nuclear magnetic resonance spectroscopy (1H-NMR). Concentrations of dietary intake biomarkers were measured with liquid chromatography triple quadrupole mass spectrometry and by integration of 1H-NMR data. Cross-validation modeling with 1H-NMR and FIE-HRMS data demonstrated the power of spot and cumulative urine samples in predicting dietary patterns in 24-h urine collections. Particularly, there was no significant loss of information when post-dinner (PD) spot or overnight cumulative samples were substituted for 24-h urine collections (classification accuracies of 0.891 and 0.938, respectively). Quantitative analysis of urine samples also demonstrated the relation between PD spot samples and 24-h urines for dietary exposure biomarkers. We conclude that PD spot urine samples are suitable replacements for 24-h urine collections. Alternatively, cumulative samples collected overnight predict similarly to 24-h urine samples and have a lower collection burden for participants.

Synthesis and Properties of C,N-Chelated Carbene Complexes of Palladium(II) with 2-Aminobenzo[d]thiazole Fragment

The reaction of bis(cyclohexylisocyanide) complex of Pd(II) with substituted benzo[d]thiazole-2-amines in the presence of triethanolamine as the base has lead to the formation of deprotonated C,N-chelated carbene complexes with a structure similar to that described previously for the products of the reaction with unsubstituted benzo[d]thiazole-2-amine. The complexes have been isolated and characterized using high-resolution mass spectrometry, IR and NMR spectroscopy (1H, 13C{1H}, 1H-1H COSY, 1H-1H NOESY, 1H-13C HSQC, 1H-13C HMBC). The resulting complexes have exhibited moderate antibacterial activity against sensitive strains of gram-negative bacteria E. coli (C600) and P. fluorescens (P218), gram-positive bacteria S. aureus (ATCC-25923) and B. subtillis (B-3142D) as well as fungi C. albicans (401/NCTC-885-653).

Resonance Assignments of Lowly Populated and Unstable Enzyme Intermediate Complex under Real-Time Conditions.

Unstable and low-abundance protein complexes represent a large family of transient protein complexes that are difficult to characterize, even by means of high-resolution NMR spectroscopy. A method to assign the NMR signals of these unstable complexes through a combination of selective isotope labeling of amino acids in a protein and site-specific labeling the protein with a paramagnetic tag is presented herein. By using this method, the resonances of unstable thioester intermediate complex (lifetime <5 h and highest concentration ≈20 μm) generated by Staphylococcus aureus sortase A and its peptide substrate under a real-time reaction have been assigned.

Hyperoxia affects the lung tissue: A porcine histopathological and metabolite study using five hours of apneic oxygenation

BackgroundOxygen is a liberally dosed medicine; however, too much oxygen can be harmful. In certain situations, treatment with high oxygen concentration is necessary, e.g. after cardiopulmonary resuscitation. The amount of oxygen and duration of hyperoxia causing pulmonary damage is not fully elucidated. The aim of this study was to investigate pathophysiological and metabolite changes in lung tissue during hyperoxia while the lungs were kept open under constant low pressure. MethodsSeven pigs were exposed to 100% oxygen for five hours, using an apneic oxygenation technique with one long uninterrupted inspiration, while carbon dioxide was removed with an interventional lung assist. Arterial blood samples were collected every 30 minutes. Lung biopsies were obtained before and after hyperoxia. Microscopy and high-resolution magic angle spinning nuclear magnetic resonance spectroscopy were used to detect possible pathological and metabolite changes, respectively. Unsupervised multivariate analysis of variance and paired sample tests were performed. A two-tailed p-value ≤ 0.05 was considered significant. ResultsNo significant changes in arterial pH, and partial pressure of carbon dioxide, and no clear histopathological changes were observed after hyperoxia. While blood glucose and lactate levels changed to a minor degree, their levels dropped significantly in the lung after hyperoxia (p ≤ 0.04). Reduced levels of antioxidants (p ≤ 0.05), tricarboxylic acid cycle and energy (p ≤ 0.04) metabolites and increased levels of several amino acids (p ≤ 0.05) were also detected. ConclusionDespite no histological changes, tissue metabolites were altered, indicating that exposure to hyperoxia affects lung tissue matrix on a molecular basis.

Poly(2-propyl-2-oxazoline)s in Aqueous Methanol: To Dissolve or not to Dissolve

At room temperature, poly(N-isopropylacrylamide) (PNIPAM) is soluble in water and methanol, but it is not soluble in certain water/methanol mixtures. This phenomenon, known as cononsolvency, has been explored in great detail experimentally and theoretically in an attempt to understand the complex interactions occurring in the ternary PNIPAM/water/co-nonsolvent system. Yet little is known about the effects of the polymer structure on cononsolvency. To address this point, we investigated the temperature-dependent solution properties in water, methanol, and mixtures of the two solvents of poly(2-cyclopropyl-2-oxazoline) (PcyPOx) and two structural isomers of PNIPAM (Mn ∼ 11 kg/mol): poly(2-isopropyl-2-oxazoline) (PiPOx) and poly(2-n-propyl-2-oxazoline) (PnPOx). The phase diagram of the ternary water/methanol/poly(2-propyl-2-oxazolines) (PPOx) systems, constructed based on cloud point (TCP) measurements, revealed that PnPOx exhibits cononsolvency in water/methanol mixtures. In contrast, methanol acts as a cosolvent for PiPOx and PcyPOx in water. The enthalpy, ΔH, and temperature, Tmax, of the coil-to-globule transition of the three polymers in various water/methanol mixtures were measured by high-sensitivity differential scanning calorimetry. Tmax follows the same trends as TCP, confirming the cononsolvency of PnPOx and the cosolvency of PiPOx and PcyPOx. ΔH decreases linearly as a function of the methanol content for all PPOx systems. Ancillary high-resolution 1H NMR spectroscopy studies of PPOx solutions in D2O and methanol-d4, coupled with DOSY and NOESY experiments revealed that the n-propyl group of PnPOx rotates freely in D2O, whereas the rotation of the isopropyl and cyclopropyl groups of PiPOx and PcyPOx, respectively, is limited due to steric restriction. This factor appears to play an important role in the case of the PPOxs/water/methanol ternary system.

Field cancerization therapy with ingenol mebutate contributes to restoring skin-metabolism to normal-state in patients with actinic keratosis: a metabolomic analysis

Actinic keratosis (AK) is a skin premalignant lesion, which progresses into squamous cell carcinoma (SCC) if left untreated. Ingenol mebutate gel is approved for local treatment of non-hyperkeratotic, non-hypertrophic AK; it also has the potential to act as a field cancerization therapy to prevent the progression of AK to SCC. To gain better insights into the mechanisms of ingenol mebutate beyond the mere clinical assessment, we investigated, for the first time, the metabolome of skin tissues from patients with AK, before and after ingenol mebutate treatment, with high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. The metabolomic profiles were compared with those of tissues from healthy volunteers. Overall, we identified a number of metabolites, the homeostasis of which became altered during the process of tumorigenesis from healthy skin to AK, and was restored, at least partially, by ingenol mebutate therapy. These metabolites may help to attain a better understanding of keratinocyte metabolism and to unmask the metabolic pathways related to cell proliferation. These results provide helpful information to identify biomarkers with prognostic and therapeutic significance in AK, and suggest that field cancerization therapy with ingenol mebutate may contribute to restore skin metabolism to a normal state in patients with AK.

Supplemental Shimming for HR-μMAS NMR Spectroscopy

Occasionally in proton high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (1H HR-MAS NMR), the standard field shimming across the region-of-interest with the active shims is not sufficient in the presence of substantial magnetic susceptibility gradients. This can be ascribed to the presence of large air pocket within the sample, the proximity between sample and probehead (especially with micro-sized probes), or data acquisition at high magnetic fields. Herein, the study demonstrates a simple approach to enhance the capacity of the shim fields—by supplementing a specific passive ferro-shim—for unifying the field distributions across the sample. Qualitative field numerical analyses were carried out to illustrate the effectiveness of the applied passive shims together with the active shims.

High-resolution pure shift NMR spectroscopy offers better metabolite discrimination in food quality analysis.

PSYCHE homonuclear decoupling, a prominent pure shift NMR method, is successfully applied to adulteration discrimination of honey and geographical originality identification of tea. Effects of homonuclear couplings are efficiently suppressed, producing resolution-enhanced spectra. The pair wise honey and tea samples are well separated in OPLS-DA models with high predictability. Due to the well-resolved and accurate assignment of singlet resonances after decoupling, PSYCHE is advantageous in the identification of differential components and accurate quantification of compound concentrations presented by enhanced volcano and Beeswarm plots of honey samples, while the analysis of NOESY is easily interfered by overlapped resonances, which is further proved by the STOCSY analysis, displaying the spectral stability and reproducibility. Experimental results show that PSYCHE can improve the spectral resolution of natural complex products such as honey and tea and be combined with multivariate statistical analysis and serve as a supplementary technique to the standard methods, especially for samples systems composed by a few high-content compounds.

Cells, drugs and NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool for investigating cellular structures and their compositions. While in vivo and whole-cell NMR have a long tradition in cell-based approaches, high-resolution in-cell NMR spectroscopy is a new addition to these methods. In recent years, technological advancements in multiple areas provided converging benefits for cellular MR applications, especially in terms of robustness, reproducibility and physiological relevance. Here, we review the use of cellular NMR methods for drug discovery purposes in academia and industry. Specifically, we discuss how developments in NMR technologies such as miniaturized bioreactors and flow-probe perfusion systems have helped to consolidate NMR's role in cell-based drug discovery efforts.

Halomethanesulfonic Acids-A New Class of Polar Disinfection Byproducts: Standard Synthesis, Occurrence, and Indirect Assessment of Mitigation Options.

Halomethanesulfonic acids (HMSAs) are recently discovered polar disinfection byproducts without commercially available reference materials. To allow for their accurate quantification, we successfully synthesized standards for the four presumably most prevalent HMSA congeners: chloromethanesulfonic acid, bromomethanesulfonic acid, dichloromethanesulfonic acid, and bromochloromethanesulfonic acid. After structure confirmation and quantification with high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, we integrated them into a multilayer solid phase extraction and hydrophilic interaction liquid chromatography-tandem mass spectrometry method dedicated to the analysis of polar water contaminants. With this method we monitored HMSAs in drinking water production plants from four European countries and tap water samples taken in six countries. HMSAs were detected in the low μg/L range after the chlorination step during drinking water production, all tap waters samples, and two surface waters used for drinking water production. Concentrations in tap water samples ranged from 0.07 μg/L to 11.5 μg/L while the HMSA concentrations in surface waters were in the range of 100 ng/L. We utilized the HMSA formation potential to indirectly assess the behavior of hitherto unknown HMSA precursors, consequently identifying ozonation, filtration through activated carbon, and reverse osmosis as efficient removal tools for HMSA precursors, thus limiting their formation during subsequent water disinfection.

Solution structural model of the complex of the binding regions of human plasminogen with its M-protein receptor from Streptococcus pyogenes

VEK50 is a truncated peptide from a Streptococcal pyogenes surface human plasminogen (hPg) binding M-protein (PAM). VEK50 contains the full A-domain of PAM, which is responsible for its low nanomolar binding to hPg. The interaction of VEK50 with kringle 2, the PAM-binding domain in hPg (K2hPg), has been studied by high-resolution NMR spectroscopy. The data show that each VEK50 monomer in solution contains two tight binding sites for K2hPg, one each in the a1- (RH1; R17H18) and a2- (RH2; R30H31) repeats within the A-domain of VEK50. Two mutant forms of VEK50, viz., VEK50[RH1/AA] (VEK50ΔRH1) and VEK50[RH2/AA] (VEK50ΔRH2), were designed by replacing each RH with AA, thus eliminating one of the K2hPg binding sites within VEK50, and allowing separate study of each binding site. Using 13C- and 15N-labeled peptides, NMR-derived solution structures of VEK50 in its complex with K2hPg were solved. We conclude that the A-domain of PAM can accommodate two molecules of K2hPg docked within a short distance of each other, and the strength of the binding is slightly different for each site. The solution structure of the VEK50/K2hPg, complex, which is a reductionist model of the PAM/hPg complex, provides insights for the binding mechanism of PAM to a host protein, a process that is critical to S. pyogenes virulence.

A pure shift and spin echo based approach for high-resolution diffusion-ordered NMR spectroscopy.

Diffusion-ordered NMR spectroscopy (DOSY) can be used for separating mixture components according to their individual diffusion behaviors, thus offering a powerful tool for the analysis of compound mixtures. However, conventional DOSY experiments generally encounter the problem of limited resolution in the spectral domain, particularly for applications to complex mixtures that contains crowed resonances in 1D NMR. In addition, chemical exchange effects, bringing about spurious component signals, pose another limitation for interpreting DOSY measurements. Here, a general DOSY method is proposed based on pure shift extraction and spin echo evolution to obtain high-resolution 2D DOSY spectra, along with the suppression on effects of chemical exchange and J coupling. Both theoretical analyses and experimental results suggest that the proposed method is useful for high-resolution DOSY measurements on complex mixtures that contains crowded or even overlapped NMR resonances and exchanging spin systems.

A novel tetraphenylethylene derivative: 4-methyl-N-[3-(1,2,2-triphenylethenyl)phenyl]benzenesulfonamide with aggregation-induced emission.

The novel tetraphenylethylene derivative 4-methyl-N-[3-(1,2,2-triphenylethenyl)phenyl]benzenesulfonamide (abbreviated as MTBF), C33H27NO2S, was synthesized successfully and characterized by single-crystal X-ray diffraction, high-resolution mass spectroscopy and 1H NMR spectroscopy. MTBF crystallizes in the centrosymmetric monoclinic space group P21/c. In the crystal structure, the MTBF molecules are connected into a one-dimensional band and then a two-dimensional sheet by hydrogen bonds of the N-H...O and C-H...O types. The sheets are further linked to produce a three-dimensional network via C-H...π interactions. The molecules aggregate via these intermolecular forces, which restrain the intramolecular motions (RIM) and decrease the energy loss in the aggregation state, so as to open the radiative channels, and thus MTBF exhibits excellent fluorescence by aggregation-induced emission (AIE) enhancement.

High Resolution Nuclear Magnetic Resonance Spectroscopy on Biological Tissue and Metabolomics.

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a universal analytical tool. It can provide detailed information on chemical shifts, J coupling constants, multiplet patterns, and relative peak areas. It plays an important role in the fields of chemistry, biology, medicine, and pharmacy. A highly homogeneous magnetic field is a prerequisite for excellent spectral resolution. However, in some cases, such as in vivo and ex vivo biological tissues, the magnetic field inhomogeneity due to magnetic susceptibility variation in samples is unavoidable and hard to eliminate by conventional methods. The techniques based on intermolecular multiple quantum coherences and conventional single quantum coherence can remove the influence of the field inhomogeneity effects and be applied to obtain highresolution NMR spectra of biological tissues, including in vivo animal and human tissues. Broadband 1H homo-decoupled NMR spectroscopy displays J coupled resonances as collapsed singlets, resulting in highly resolved spectra. It can be used to acquire high-resolution spectra of some pharmaceuticals. The J-difference edited spectra can be used to detect J coupled metabolites, such as γ-aminobutyric acid, the detection of which is interfered by intense neighboring peaks. High-resolution 1H NMR spectroscopy has been widely utilized for the identification and characterization of biological fluids, constituting an important tool in drug discovery, drug development, and disease diagnosis.

4-Imidazol-1-yl-butane-1-sulfonic acid ionic liquid: Synthesis, structural analysis, physical properties and catalytic application as dual solvent-catalyst

4-Imidazol-1-yl-butane-1-sulfonic acid (ImBu-SO3H) has been successfully synthetized and fully characterized by FT-IR and high-resolution NMR spectroscopy (1H, 13C). The “plausible” alternative structures of ImBu-SO3H were discussed on the basis of its NMR data. The ionic liquid showed interesting dual solvent-catalyst property, which was studied experimentally for the acetylation of a variety of functionalized alcohols, phenols, thiols, amines and α-tocopherol (α-CTP) as the most active form of vitamin E with acetic anhydride and which provided good yields within a short reaction time. ImBu-SO3H was successfully recycled by product extraction with an average recovered yield of 82% for 5 subsequent runs. The catalytic activity of the recycled ImBu-SO3H showed almost no loss even after five consecutive runs.

Tracking the precession of single nuclear spins by weak measurements.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for analysing the structure and function of molecules, and for performing three-dimensional imaging of their spin densities. At the heart of NMR spectrometers is the detection of electromagnetic radiation, in the form of a free induction decay signal1, generated by nuclei precessing around an applied magnetic field. Whereas conventional NMR requires signals from 1012 or more nuclei, recent advances in sensitive magnetometry2,3 have dramatically lowered the required number of nuclei to a level where a few or even individual nuclear spins can be detected4-6. It is unclear whether continuous detection of the free induction decay can still be applied at the single-spin level, or whether quantum back-action (the effect thata detector has on the measurement itself) modifies or suppresses the NMR response. Here we report the tracking of single nuclear spin precession using periodic weak measurements7-9. Our experimental system consists of nuclear spins in diamond that are weakly interacting with the electronic spin of a nearby nitrogen vacancy centre, acting as an optically readable meter qubit. We observe and minimize two important effects of quantum back-action: measurement-induced decoherence10 and frequency synchronization with the sampling clock11,12. We use periodic weak measurements to demonstrate sensitive, high-resolution NMR spectroscopy of multiple nuclear spins with a priori unknown frequencies. Our method may provide a useful route to single-molecule NMR13,14 at atomic resolution.

Magnesium and calcium reveal different chelating effects in a steroid compound: A model study of prednisolone using NMR spectroscopy

In this work, we used high resolution NMR spectroscopy to investigate metal cation chelation by the steroidal drug 1,4-pregnadiene-11β,17α,21-triol-3,20-dione (Prednisolone; abbreviated as Prd). Prd/MgCl2 and Prd/CaCl2 mixtures were prepared at eight different molar ratios. Using two-dimensional 1H/13C heteronuclear correlation spectroscopy, we were able to resolve most of the 1H signals, except those at 1.4–1.55 ppm, where signals for H15β, H16α and Me-19 are superimposed. The chelation sites were determined by the cation concentration-dependent 13C signals. Both ring A and ring D of Prd were found to be involved in Mg2+ chelation, whereas only ring A was involved in Ca2+ chelation. The dihedral angles deduced from the 3JH-H coupling constants indicated that ring D of Prd might undergo rather small, but different, distortions in the presence of Mg2+ and Ca2+. Additionally, using the continuous variation method, we deduced that the stoichiometric ratios of the Prd/Mg2+ and Prd/Ca2+ complexes were 1:1 and 2:1, respectively. All of the evidence led us to conclude that the Prd/Mg2+ and Prd/Ca2+ complexes are mediated by different chelating mechanisms.

Comparative analysis of petroleum and coal pitches using 1H and 13C NMR spectroscopy

Petroleum and coal pitches are known to be among the most important sources of raw materials for the production of carbon materials, including electro-carbon, heat- and chemical-resistant structural products, metal-carbon and carbon-carbon composite materials, graphite electrodes, self-baking anode paste, carbon fibres, blast furnace and cupola coke. The quality of the pitches is determined by the elemental and group composition, as well as their structural and physical-chemical properties. The study of the molecular structure and group composition of the organic components of the pitch and the identification of the effect of the composition on the performance of the products is of great interest for assessing the prediction of the behaviour of pitches during processing and the properties of the products obtained from them. The present work is devoted to the study of the group composition of two petroleum and two coal pitches using high-resolution NMR spectroscopy. The data of the ¹H and ¹³C NMR spectra allow the composition of the pitch product to be estimated without separation into fractions, which, in turn, combined with the accuracy of the method and the recording speed of the ¹H NMR spectra, increases the rapidity of this method of analysis. The combination of ¹N and ¹³C NMR data allows additional information on the correlation of physical parameters and pitch composition to be established. The calculation of the (H ar /H al ) parameter, corresponding to the ratio of the integral intensities of signals of aromatic and aliphatic hydrogen atoms, provides the possibility of using it as a structural characteristic for a particular sample. In order to ensure the correct contribution to the integral intensity of the signals, ¹³C NMR spectra were recorded in a pulse sequence with suppression of the spin-spin proton interaction only for the period of data reading for the purposes of minimising the nuclear Overhauser effect. The relaxation delay between pulses was set to 10 s. The study of petroleum and coal pitches using NMR spectroscopy showed the proton spectra of NMR data to be sufficiently informative and suitable for monitoring the technological process of pitch production.