1H-13C HSQC Spectra Research Articles

Identifying Anti-NSCLC Bioactive Compounds in Scutellaria via 2D NMR-Based Metabolomic Analysis of Pharmacologically Classified Crude Extracts.

We presented a strategy utilizing 2D NMR-based metabolomic analysis of crude extracts, categorized by different pharmacological activities, to rapidly identify the primary bioactive components of TCM. It was applied to identify the potential bioactive components from Scutellaria crude extracts that exhibit anti-non-small cell lung cancer (anti-NSCLC) activity. Four Scutellaria species were chosen as the study subjects because of their close phylogenetic relationship, but their crude extracts exhibit significantly different anti-NSCLC activity. Cell proliferation assay was used to assess the anti-NSCLC activity of four species of Scutellaria. 1H-13C HSQC spectra were acquired for the chemical profiling of these crude extracts. Based on the pharmacological classification (PCA, OPLS-DA and univariate hypothesis test) were performed to identify the bioactive constituents in Scutellaria associated with the anti-NSCLC activity. As a result, three compounds, baicalein, wogonin and scutellarin were identified as bioactive compounds. The anti-NSCLC activity of the three potential active compounds were further confirmed via cell proliferation assay. The mechanism of the anti-NSCLC activity by these active constituents was further explored via flow cytometry and western blot analyses. This study demonstrated 2D NMR-based metabolomic analysis of pharmacologically classified crude extracts to be an efficient approach to the identification of active components of herbal medicine.

INCREASING THE EFFICIENCY OF USING WOOD BIOMASS BY PROVIDING SPECIAL PROPERTIES

Background. The search for ways to use logging waste is a distinct scientific direction, due to the large number of their permanent formation. The importance of such studies is associated with the need to reduce the loss of valuable wood raw materials in the form of logging waste. One of the possible ways of using waste from logging industries is the production of wood chips using a mobile complex of machines with its further recycling into a wood-fiber semi-finished product. Wood-fiber semi-finished product is, as it is known nowadays, one of the environmentally friendly types of insulation and finishing materials. A significant limitation in the use of wood fiber in various types of production is low fire and bio-resistance. As a consequence, imparting specific properties to a wood-fiber semi-finished product in the form of fire and bio-resistance is an acute problem of the modern wood processing industry.
 Purpose. Synthesis of 4-azofunctionalized pyrazoles with perfluoroalkyl substituent by cyclocondensation of polyfluorinated beta-diketones to confer special properties to the wood-fiber semi-finished product.
 Materials and methods. The 1H, 13C NMR and 1H-13C HSQC spectra were registered at the Krasnoyarsk Regional Center for Collective Use of the FITC KNC SB RAS on a Bruker Avance III Fourier spectrometer with a frequency of 600 MHz equipped with sensitive helium probes with a diameter of 5 mm and 1.7 mm. TMS is used as an internal standard. Chromato-mass spectra were obtained using ISQ 7610 Single Quadrupole GC-MS and Shimadzu LC/MS-2020 instruments. Methyl alcohol was used as the mobile phase. IR spectra were recorded on the FT-801 (FT-801) SIMEX IR Fourier spectrometer with a spectral range from 450 to 5700 cm-1. UV spectra were recorded on a HELIOS OMEGA spectrophotometer equipped with a tungsten-halogen lamp in quartz cuvettes with a diameter of 1 cm at a concentration of 1·10-4 mol/l for 200-400 nm and a concentration of 1·10-2 mol/l for 400-800 nm in ethanol. Melting temperatures were determined in open capillaries on a PTP device (TU 25-11-1144-76).
 Results. 3 new compounds were synthesized: 3(5)-(thiophene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole, 3(5)-phenyl-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole and 3(5)-(naphthalene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole.
 Conclusion. Thus, 3(5)-(thiophene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole, 3(5)-phenyl-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole and 3(5)-(naphthalene-2-yl)-4-nitroso-5(3)-(trifluoromethyl)-1H-pyrazole. Complex separable mixtures of substances containing pyrazoline, β-diketone and target nitrosopyrazoles were formed by a single-reactor method. The two-stage method of production proposed by us increases the purity and yield of the target products.

A novel phenanthroline[9,10-d] imidazole-based fluorescent sensor for Hg2+ with “turn-on” fluorescence response

Herein, a phenanthro[9,10-d] imidazole Schiff based “turn-on” fluorescence sensor (compound 1) was synthesized by 4-hydroxy-3-nitrobenzaldehyde and 9,10-phenanthrenequinone. In DMF/PBS buffer solution (1:4, v/v, pH 7.4), compound 1 responsed to Hg2+ with high sensitivity (detection limit was 45.76 nM) and rapid response (completed within 5 s), accompanied by the fluorescence change from null to blue. The binding ratio of compound 1 to Hg2+ was 1:1, and the binding constant was 2.933 × 106 M−1. Through detailed analysis of 1H–1H COSY and 1H–13C HSQC spectra, we proposed the possible fluorescence emission mechanism of compound 1 and Hg2+, and verified the recognition mechanism of compound 1 through density functional theory (DFT) calculation. Compound 1 was successfully used to detect Hg2+ in actual water samples, the recovery was 91 %–106 %, and the relative standard deviation (R.S.D.) was less than 2.5 %, which provided possibility for compound 1 to be used as a sensor in environmental research.

Unambiguous Identification of Glucose-Induced Glycation in mAbs and other Proteins by NMR Spectroscopy

ObjectiveGlycation is a non-enzymatic and spontaneous post-translational modification (PTM) generated by the reaction between reducing sugars and primary amine groups within proteins. Because glycation can alter the properties of proteins, it is a critical quality attribute of therapeutic monoclonal antibodies (mAbs) and should therefore be carefully monitored. The most abundant product of glycation is formed by glucose and lysine side chains resulting in fructoselysine after Amadori rearrangement. In proteomics, which routinely uses a combination of chromatography and mass spectrometry to analyze PTMs, there is no straight-forward way to distinguish between glycation products of a reducing monosaccharide and an additional hexose within a glycan, since both lead to a mass difference of 162 Da.MethodsTo verify that the observed mass change is indeed a glycation product, we developed an approach based on 2D NMR spectroscopy spectroscopy and full-length protein samples denatured using high concentrations of deuterated urea.ResultsThe dominating β-pyranose form of the Amadori product shows a characteristic chemical shift correlation pattern in 1H-13C HSQC spectra suited to identify glucose-induced glycation. The same pattern was observed in spectra of a variety of artificially glycated proteins, including two mAbs, as well as natural proteins.ConclusionBased on this unique correlation pattern, 2D NMR spectroscopy can be used to unambiguously identify glucose-induced glycation in any protein of interest. We provide a robust method that is orthogonal to MS-based methods and can also be used for cross-validation.

Introducing Molecular Structural Analysis Using a Guided Systematic Approach Combined with an Interactive Multiplatform Web Application

Performing the identification of organic chemical compounds from a set of spectroscopic data gives the opportunity to students to develop critical thinking and problem-solving skills. In this context, we developed a student-centered methodology for teaching molecular structural analysis to first-year undergraduate students. This systematic approach was implemented during classroom-training sessions and complemented by a home-based training program. Home-based activities involved a multiplatform Web-based application (ULg Spectra) combined with guided inquiries. ULg Spectra offers fully interactive tutorial/drill materials relying on mass, infrared, and nuclear magnetic resonance spectra (i.e., one-dimensional 1H NMR and 13C NMR spectra and two-dimensional 1H–13C HSQC spectra). A survey indicates that the vast majority of students valued the ULg Spectra application combined with guided inquiries, especially in terms of usability and usefulness. This approach prompted them to actively engage in problem solving, and student autonomy was improved. Statistical data demonstrated that low-, medium-, and high-training students’ groups showed increasing performance in the final exam. Interestingly, a statistically significant increase in final grades and success rate was also observed compared to previous years.

Conjugates of Polyguluronic and Polymannuronic Acids with 4-Aminoantipyrine. Determination Using 1H–13C HSQC Spectra of Triad Units and Their Distribution in the Polysaccharide Chain

Conjugates of poly-α-(1→4)-L-guluronic and poly-β-(1→4)-D-mannuronic acids with 1-phenyl-2,3-dimethyl-4-aminopyrazol-5-one (4-aminoantipyrine) were synthesized and characterized using 1H–13C HSQC spectra by the contents of intact A units, modified B units, and triad sequences of units with an A center (AAA, AAB + BAA, and BAB) and with a B center (BBB, BBA + ABB, and ABA).

Investigation of the Influence Glycosaminoglycan Sulfation on the Interaction with Interleukin-8 by Fluorescence and Solution NMR Spectroscopy

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix (ECM), and proteins such as growth factors and chemokines play a critical role in cellular regulation processes. Previous work on the chemokine Interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. Nevertheless, low or non sulfated GAGs such as hyaluronan (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), also contained in the extracellular matrix, have so far not been studied with regard to their distinct binding properties towards IL-8.In this work, we combine fluorescence as well as solution NMR experiments to study the recognition properties of GAG hexasaccharides, including HA, CS, DS and heparin, to IL-8. Using 1H-15N HSQC spectroscopy of IL-8 in the presence of varying GAG concentrations, the interacting amino acid could be identified. Furthermore to observe lysine side chains, a reductive methylation with [13C]-formaldehyde, adding two methyl groups to the e-NH3+ of lysines without altering the positive charge, was performed. In order to resolve the signals from the lysine side chains, 1H-13C HSQC spectra were recorded and the influence of the GAGs was tested again.The results show that an increase in GAG sulfation enhances the strength of the binding to the protein. In addition, in cases of equal degree of sulfation the position of the sulfate group plays a crucial role sugessting that interactions between proteins and GAGs are not purely electrostatically driven and steric forces as well as contributions from hydrogen bonds have to be considered. These observations were also confirmed by molecular docking and dynamic simulations providing a structural picture of the interaction of IL-8 with various GAGs.

Structural Investigations of ClC-ec1, a Large Integral Membrane Protein, using Solution-State NMR and Nanodisc Technology

CLC antiporters mediate Cl-/H+ exchange across cell membranes in organisms ranging from bacteria to mammals. This exchange is accomplished through elegant coupling of protein conformational changes to ion binding, unbinding and translocation events. To understand this mechanism, molecular details of the different conformations and the dynamics of their interchange must be known. While X-ray crystallographic structures have provided essential molecular pictures, the details of protein conformational change have remained elusive. To address this issue, we are using solution-state NMR to probe conformational change in ClC-ec1, a well-characterized CLC homolog of known structure. Selective 13C labeling of lysine residues and the N-terminus is achieved by post-translational reductive methylation. 1H-13C HSQC spectra of these samples reveal reversible, substrate-dependent spectral changes that may reflect protein conformational changes (studies in progress). A chronic concern with the use of detergent-solubilized protein is the possible effects of the non-native detergent environment. To address this concern we are also implementing nanodisc technology. Nanodiscs present a promising alternative for studying membrane proteins such as CLCs, offering both a native bilayer environment and a size small enough for solution-state NMR studies. Each disc consists of two amphipathic helices wrapped around a bilayer patch of lipids surrounding the membrane-embedded protein. The protocol for the incorporation of the monomeric, 13C-methylated mutant form of ClC-ec1 was optimized and the samples tested for homogeneity and monodispersity. The 1H-13C HSQC spectra of methylated, monomeric ClC-ec1 in nanodiscs are being investigated, with preliminary results indicating substrate-dependent spectral changes in this system. While further experiments will be needed to determine if these spectral changes represent functionally-relevant conformational changes, these studies demonstrate the potential for using solution-state NMR and nanodisc technology to study ClC-ec1 structure in a native lipid environment.

Determination of the relative pKa's of mixtures of organic acids using NMR titration experiments based on aliased 1H–13C HSQC spectra

NMR titrations can be used to simultaneously determine the pKa's of mixtures of organic acids and bases provided chemical shifts are sensitive to their change of protonation states. Carbons are in principle better probes than protons because their chemical shifts are typically more sensitive to the changes in the electronic densities of the functional groups they are bound to. They are also less prone to overlap, do not suffer from second-order effects and are less sensitive to changes in solvent conditions. Recording HSQC spectra satisfactorily reduces the lack of sensitivity of carbon-detected experiments and the signal dispersion in two dimensions allows to automatically and finely track the drifts of a large number of signals simultaneously. The application of spectral aliasing to the carbon dimensions allows to quickly reach the high resolution required to precisely measure variations in carbon chemical shifts. The paper extends the methodology to the treatment of the data stemming from the simultaneous analysis of mixtures of compounds. It also introduces methods to improve the normalization process and deal with missing points. The application is not limited to the determination of pKa and can be readily extended to the determination of other chemical constants in experiments where the components of the mixture dynamically compete for substrates other than protons.

Using NMR Metabolomics to Investigate Tricarboxylic Acid Cycle-dependent Signal Transduction in Staphylococcus epidermidis

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.

Rapid determination of antigenic epitopes in human NGAL using NMR

The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two-dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high-affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase-associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in (1)H-(15)N-TROSY HSQC and (1)H-(13)C HSQC spectra of isotope-labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual-color fluorescence cross-correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel-like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR-based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest.

Structure of the Calcineurin-NFAT Complex: Defining a T Cell Activation Switch Using Solution NMR and Crystal Coordinates

Calcineurin (Cn) is a serine/threonine protein phosphatase that plays pivotal roles in many physiological processes, including cell proliferation, development, and apoptosis. Most prominently, Cn targets the nuclear factors of activated T cell (NFATs), transcription factors that activate cytokine genes. Calcium-activated Cn dephosphorylates multiple residues within the regulatory domain of NFAT, triggering joint nuclear translocation. This relies crucially on the interaction between the catalytic domain of Cn (CnCat) and the conserved PxIxIT motif located in a region distinct from the dephosphorylation sites of NFAT. Here, we present the structure of the complex between the 39 kDa CnCat and a 14 residue peptide containing a PVIVIT segment that was derived from affinity-driven peptide selection based on the conserved PxIxIT motif of NFATs. The structure of the complex was determined by using NMR assignments and structural constraints and the coordinates of the CnCat crystal structure. The NMR analysis relied on recently developed labeling and spectroscopic techniques. The VIVIT peptide is accommodated in a hydrophobic cleft formed by beta strands 11 and 14, and the loop between beta strands 11 and 12, forming a short parallel beta sheet with the exposed beta strand 14 in Cn. The side chains of conserved residues in the PxIxIT sequences make extensive interactions with conserved residues in Cn, while those of nonconserved residues are solvent exposed. The architecture of the interface explains the diversity of recognition sequences compatible with NFAT function and uncovers a potential targeting site for immune-suppressive agents. The structure reveals that the orientation of the bound PxIxIT directs the phosphorylation sites in NFAT's regulatory domain toward the Cn catalytic site.

Characterization of the malate‐aluminum(III) complex using 1H and 27Al NMR spectroscopy

Structural elucidation of a malate-aluminum(III) complex has been carried out using 1H and 27Al NMR spectroscopy. The 1H chemical shift perturbation clearly indicated the interaction between malate and Al(III) ion. The measurements of 27Al NMR and 1H-13C HSQC spectra demonstrated that the major form of a complex comprised two equivalent malate ions and two unequivalent Al(III) ions. With this constraint, an equilibrium geometry of the complex was proposed by a semi-empirical molecular orbital calculation.

Ultrahigh-Resolution 1H−13C HSQC Spectra of Metabolite Mixtures Using Nonlinear Sampling and Forward Maximum Entropy Reconstruction

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultrahigh-resolution 1H-13C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time-consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using nonlinear sampling (NLS) and a new high-fidelity forward maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time-domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time-domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points, it reproduces signal intensities with high fidelity and does not suffer from a dynamic range problem. As an example we measured a natural abundance 1H-13C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1H-13C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from one-seventh of the increments with nearly identical spectral appearance, indistinguishable signal intensities, and comparable or even lower root-mean-square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultrahigh resolution 1H-13C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins.

A simple spectral editing procedure is described that generates separate subspectra for the methyl 13C-[1H3] multiplet components of 1H-13C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields 1H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of 1H-13C J and dipolar couplings. The editing procedure is illustrated for a Ca2+-calmodulin sample in isotropic and liquid crystalline phases.

Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.