Re-Evaluation of Binding Properties of Recombinant Lymphocyte Receptors NKR-P1A and CD69 to Chemically Synthesized Glycans and Peptides

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-d-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.     

Structure induction of the T-cell receptor zeta-chain upon lipid binding investigated by NMR spectroscopy

The conformation of the cytoplasmic part of the zeta-chain of the T-cell receptor (TCR) in its free form and bound to detergent micelles has been investigated by heteronuclear NMR spectroscopy. The zeta-chain is considered to be a mediator between the extracellular antigen and the intracellular signal-transduction cascade leading to T-cell activation. Earlier studies suggested a T-cell activation mechanism that involved a TCR-state-dependent lipid incorporation propensity of the zeta-chain accompanied by a helical folding transition. In order to support this proposed mechanism, standard protein NMR assignment and secondary-structure-elucidation techniques have been applied to the free TCR zeta-chain and to the zeta-chain bound to the detergent LMPG, which forms a micelle, in order to obtain the structural characteristics of this folding transition in a residue-resolved manner. We could assign the resonances of the free zeta-chain at 278 K, and this formed the basis for chemical-shift-perturbation studies to identify lipid binding sites. Our NMR results show that the free TCR zeta-chain is indeed intrinsically unstructured. Regions around the ITAM2 and ITAM3 sequences are involved in a highly dynamic binding of the free zeta-chain to a detergent micelle formed by the acidic lipid LMPG.     

Determination of crosslink densities of carbon black filled natural rubber sulfur vulcanizates by 13C NMR MAS spectroscopy and 1H NMR transversal relaxation

Two NMR spectroscopic methods were applied to carbon black filled NR vulcanizates to determine the chemical crosslink density. Both methods give corresponding results. Addition of carbon black leads to a decrease of crosslink density with increasing filler content. The influence is more pronounced at higher sulfur content.     

Determination of the fatty acid profile by 1H‐NMR spectroscopy

The common unsaturated fatty acids present in many vegetable oils (oleic, linoleic and linolenic acids) can be quantitated by ¹H‐nuclear magnetic resonance spectroscopy (¹H‐NMR). A key feature is that the signals of the terminal methyl group of linolenic acid are shifted downfield from the corresponding signals in the other fatty acids, permitting their separate integration and quantitation of linolenic acid. Then, using the integration values of the signals of the allylic and bis‐allylic protons, oleic and linoleic acids can be quantitated. The procedure was verified for mixtures of triacylglycerols (vegetable oils) and methyl esters of oleic, linoleic and linolenic acids as well as palmitic and stearic acids. Generally, the NMR (400 MHz) results were in good agreement with gas chromatographic (GC) analyses. As the present ¹H‐NMR‐based procedure can be applied to neat vegetable oils, the preparation of derivatives for GC would be unnecessary. The present method is extended to quantitating saturated (palmitic and stearic) acids, although in this case the results deviate more strongly from actual values and GC analyses. Alternatives to the iodine value (allylic position equivalents and bis‐allylic position equivalents) can be derived directly from the integration values of the allylic and bis‐allylic protons.     

1H NMR spectroscopy as tool to follow changes in the fatty acids of fish oils

High‐resolution NMR spectroscopy is a fast and accurate tool to measure the content of n‐3 PUFA in unoxidised oils. Four types of fish oils with a high amount of n‐3 PUFA were oxidised under different conditions, and the changes in their fatty acid pattern were monitored with NMR spectroscopy and compared with traditional analytical methods, especially GC. Good compliances were obtained for unoxidised oils and for oils at the early oxidation stages. Distinct changes in signals or signal ratio intensities arose only in higher‐oxidised oils that were irradiated or heated in the presence of oxygen over several days. Slight differences between values obtained by GC and NMR at this point were likely caused by accumulation of oxidation products. Detection of the terminal lipid oxidation products propanal and 2‐propenal was only possible at late oxidation stages.     

The relationship of adsorbed species observed by infrared spectroscopy to the mechanism of ethylene oxidation over silver

A combined infrared and kinetic investigation was performed on the oxidation of ethylene over a silver catalyst. Based upon the surface structures deduced from the spectra and additional information taken from the literature, a mechanism was developed to interpret both the epoxidation and combustion processes. The proposed mechanism explains qualitatively the kinetics observed in the present investigation and the variations in selectivity as a function of reactant and product concentrations reported in the literature by previous investigators.     

Binding of the globular domain of linker histones H5/H1 to the nucleosome: a hypothesis

The amino acid sequence of the central globular domain of histoneH1/H5 family members is highly homologous. Twenty-four suchsequences have been compared to establish the conserved andvariable residues. Fitting this to the tertiary structure ofthe H5 globular domain shows which of the conserved and variableresidues are peripheral and which internal. Particular attentionis paid to conserved basic residues on the surface, which wetake to be DNA binding. Variable regions and conserved acidicresidues are assumed not to be sites of contact with DNA. Weconclude that one face of the domain, containing a cluster ofbasic residues, is the principal DNA binding site whilst twoopposing faces, orthogonal to the principal site and also containingconserved basic residues, are subsidiary DNA binding sites.Since the DNA binding surface of the domain covers a full 180°arc, we propose that it contacts a ‘cage’ of threeDNA strands on the 2-fold axis of the chromatosome.     

A study of the structure of poly(hexene-1) prepared by nickel(α-diimine)/MAO catalyst using high resolution NMR spectroscopy

Homopolymerization of hexene-1 with nickel(α-diimine) catalyst forms poly(hexene-1) containing different types of branches and varying number of methylene units in the backbone. Types of branches identified by NMR spectroscopy include, butyl (C₄), longer than butyl (>C₄) and methyl (isolated and meso and racemic head-to-head). Formation of ethyl (C₂) and propyl (C₃) branches were not observed. The migration of the nickel during polymerization not only causes the formation of branches other than C₄, but also the runs of methylene units in the backbone due to complete 1,6-enchainment (chain running). The formation of regio-irregular methyl branches required an insertion of hexene-1 in the nickel-secondary carbon. Formation of methyl branches and (1, ω) enchainment has also been observed during the polymerization of octene-1, decene-1, tetradecene-1 using the same catalyst system. The extent formation of these subunits in the polymer depends on polymerization temperature. A plausible explanation for the formation of different branches is discussed.     

Studies on the radical polymerization of vinyl acetate in benzene,chlorobenzene and ethyl acetate by 1H NMR spectroscopy

Summary Polymerizations of vinyl acetate were carried out with AIBN in benzene, chlorobenzene and ethyl acetate, and the resultant polymers were analyzed for terminal group by using ¹H NMR technique. The results revealed that a part of the polymer molecules prepared in aromatic solvent contained one solvent fragment at the chain end, indicating the incorporation of aromatic molecule through the chain transfer reaction and not by the copolymerization. It was found that there existed at least 0.7 branching per a molecule in the polymer prepared in ethyl acetate, whereas almost no branching in the polymer prepared in aromatic solvents.     

Monitoring the grafting of epoxidized natural rubber by size‐exclusion chromatography coupled to FTIR spectroscopy

The evaluation of heterogeneous polymeric species by a selective, dual detector size‐exclusion chromatography setup can provide accurate results on the incorporation of specific functional groups in copolymers as a function of the molar mass distribution. However, when non‐UV‐absorbing species are used in copolymerization reactions, the dual detector method becomes less reliable. By interfacing a Fourier transform infrared (FTIR) spectrometer with size‐exclusion chromatography (SEC), the problem can be overcome, making it possible to map non‐UV‐absorbing species as a function of the molar mass distribution. Coupling takes place via a solvent‐evaporation stage, which delivers the mobile phase as a dry, solvent‐free polymeric film onto a germanium disk. In this article, styrene and methyl methacrylate were grafted onto epoxidized natural rubber (ENR50) and analyzed by SEC. The accuracy of FTIR as a suitable detector was evaluated by comparing results from a dual detector SEC setup and FTIR coupled to SEC. FTIR proved to be a successful detector for the analysis of non‐UV‐absorbing species. This was consequently followed by the characterization of methyl methacrylate‐grafted ENR50. From the relevant data, Gram–Schmidt and contour plots could be made to indicate the incorporation of methyl methacrylate into the grafted epoxidized natural rubber as a function of the molar mass distribution. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2539–2549, 2003     

pH effects on the conformational preferences of amyloid beta-peptide (1-40) in HFIP aqueous solution by NMR spectroscopy

The structure and aggregation state of amyloid beta-peptide (Abeta) in membrane-like environments are important determinants of pathological events in Alzheimer's disease. In fact, the neurotoxic nature of amyloid-forming peptides and proteins is associated with specific conformational transitions proximal to the membrane. Under certain conditions, the Abeta peptide undergoes a conformational change that brings the peptide in solution to a "competent state" for aggregation. Conversion can be obtained at medium pH (5.0-6.0), and in vivo this appears to take place in the endocytic pathway. The combined use of (1)H NMR spectroscopy and molecular dynamics-simulated annealing calculations in aqueous hexafluoroisopropanol simulating the membrane environment, at different pH conditions, enabled us to get some insights into the aggregation process of Abeta, confirming our previous hypotheses of a relationship between conformational flexibility and aggregation propensity. The conformational space of the peptide was explored by means of an innovative use of principal component analysis as applied to residue-by-residue root-mean-square deviations values from a reference structure. This procedure allowed us to identify the aggregation-prone regions of the peptide.     

Analysis of biodiesel,diesel and gasoline by NMR spectroscopy – A quick and robust alternative to NIR and GC

¹H NMR spectroscopy is presented as a routine analysis for diesel mixed with biodiesel and gasoline. Results of NMR spectroscopy and the orthogonal gas chromatography method show identical results.     

Investigation of microstructure of the acrylonitrile‐styrene‐glycidyl methacrylate terpolymers by 1D and 2D NMR spectroscopy

Acrylonitrile‐styrene‐glycidyl methacrylate (N/S/G) terpolymers were prepared by bulk polymerization by using benzoyl peroxide as initiator and analyzed by NMR spectroscopy. The compositions of terpolymers were determined by quantitative ¹³C{¹H}‐NMR spectra and compared with those calculated by Goldfinger's equation by using comonomer reactivity ratios: r_NS = 0.04, r_SN = 0.40; r_NG = 0.22, r_GN = 1.37; r_SG = 0.44, r_GS = 0.53. The ¹³C{¹H}‐ and ¹H‐NMR spectra were overlapping and complex. The spectral assignments were done with the help of distortionless enhancement by polarization transfer and two‐dimensional ¹³C‐¹H heteronuclear single quantum correlation experiments. 2D total correlated spectroscopy was used to ascertain the various coupling between the protons. The methyl, methine, methylene, and oxymethylene carbon resonances showed compositional sensitivity. 2D nuclear Overhauser enhancement spectroscopy (NOESY) experiment was used to ascertain the spatial proton–proton couplings. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1779–1790, 2003     

Determination of optical purity and absolute configuration of natural 12‐hydroxystearic acid by the 1H NMR anisotropy method

The enantiopure of (R)‐(?) MαNPA was allowed to react with racemic 18‐(tert‐butyldimethylsilyloxy)‐5‐octadecayne‐7‐ol which was derived from dodecane‐1,12‐diol, yielding diastereomeric esters mixture. These racemic esters were easily separated by HPLC on silica‐gel. The absolute configurations of the first‐eluted diastereomeric esters from the separated esters were determined using ¹H NMR anisotropy method. Analysis on ¹H NMR spectra and HPLC elution time of the synthesized esters and those of MαNP ester derived from natural methyl‐12‐hydroxystealate showed that the absolute configuration of natural 12‐hydroxystearic acid was R form, and the enantiomeric excess was over 99%.     

Kinetic study of the free‐radical polymerization of vinyl acetate in the presence of deuterated chloroform by 1H‐NMR spectroscopy

The free‐radical polymerization of vinyl acetate was performed in the presence of deuterated chloroform (CDCl₃) as a chain‐transfer agent (telogen) and 2,2′‐azobisisobutyronitrile as an initiator. The effects of the initiator and solvent concentrations (or equivalent monomer concentration) and the reaction temperature on the reaction kinetics were studied by real‐time ¹H‐NMR spectroscopy. Data obtained from analysis of the ¹H‐NMR spectra were used to calculate some kinetic parameters, such as the initiator decomposition rate constant (k_d), k_p(f/k_t)^1/2 ratio (where k_p is the average rate constant for propagation, f is the initiator efficiency, and k_t is the average rate constant for termination), and transfer constant to CDCl₃ (C). The results show that k_d and k_p(f/k_t)^1/2 changed significantly with the solvent concentration and reaction temperature, whereas they remained almost constant with the initiator concentration. C changed only with the reaction temperature. Attempts were made to explain the dependence of k_p(f/k_t)^1/2 on the solvent concentration. We concluded from the solvent‐independent C values that the solvent did not have any significant effect on the k_p values. As a result, changes in the k_p(f/k_t)^1/2 values with solvent concentration were attributed to the solvent effect on the f and/or k_t values. Individual values of f and k_t were estimated, and we observed that both the f and k_t values were dependent on the solvent (or equivalent monomer) concentration. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best-characterized protein kinases is the catalytic subunit of cAMP-dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply ((2)H,(13)C,(15)N) labeled protein and classical NMR assignment experiments, 2) back-calculation of chemical shifts from known X-ray structures, 3) use of paramagnetic adenosine derivatives as spin-labels, and 4) selective amino acid labeling. Interpretation of chemical-shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D (1)H,(15)N TROSY of an inactive Thr197Ala mutant with the wild-type enzyme.     

Kinetic study of radical polymerization. VII. Investigation into the solution copolymerization of acrylonitrile and itaconic acid by real‐time 1H NMR spectroscopy

Free radical solution copolymerization of acrylonitrile (AN) and itaconic acid (IA) was performed with DMSO‐d₆ as the solvent and 2,2′‐azobisisobutyronitrile (AIBN) as the initiator. Weight ratio of the monomers to solvent and molar ratio of initiator to monomers were constant in all experiments. The initial comonomer composition was the only variable in this study. On‐line ¹H NMR spectroscopy was applied to follow individual monomer conversion. Mole fraction of AN and IA in the reaction mixture (f) and in the copolymer chain (F) were measured with progress of the copolymerization reaction. Overall monomer conversion versus time and also compositions of monomer mixture and copolymer as a function of overall monomer conversion were calculated from the data of individual monomer conversion versus time. Total rate constant for the copolymerization reaction was calculated by using the overall monomer conversion versus time data and then k_p/k_t^0.5 was estimated. The dependency of k_p/k_t^0.5 on IA concentration was studied and it was found that this ratio decreases by increasing the mole fraction of IA in the initial feed. The variation of comonomer and copolymer compositions as a function of overall monomer conversion was calculated theoretically by the terminal model equations and compared with the experimental data. Instantaneous copolymer composition curve showed the formation of alternating copolymer chain during copolymerization reaction. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3253–3260, 2007