Methyl Sites Research Articles

Measurement of Methyl 2H Quadrupolar Couplings in Oriented Proteins. How Uniform Is the Quadrupolar Coupling Constant?

2H quadrupolar and one-bond 13C−13C dipolar couplings have been measured at methyl sites in a uniformly 13C and fractionally 2H labeled sample of the N-terminal drk SH3 domain, weakly aligned in a dilute solution of Pf1 phage. An average ratio between the 2H quadrupolar and 13C−13C dipolar couplings of 19.2 ± 0.1 is measured. Assuming rapid rotation about the one-bond 13Cmethyl−13C axis and an angle of 109.5° between the unique principal axis of the electric field gradient tensor and the methyl averaging axis (13Cmethyl−13C bond), an average value of 167 ± 1 kHz is obtained for the quadrupolar coupling constant, e2qQ/h. The profile of 2H quadrupolar vs 13C−13C dipolar couplings suggests that the use of a uniform value for the quadrupolar coupling constant is a good approximation in the analysis of 2H relaxation data measured at methyl sites in proteins.

Stable isotope characterization of milk components and whey ethanol.

A multi-isotopic study of several components of milk has been carried out on commercial samples and on milk produced in feeding experiments involving different kinds of diets originating from C(3) or C(4) photosynthetic metabolisms and exhibiting a relatively wide range of isotope ratios. The dispersion of the carbon, hydrogen, and nitrogen isotope parameters of dried matter and of the lactose, protein, and lipid components has been estimated. In addition, the carbohydrates were represented by the site specific isotope ratios (SNIF-NMR) of ethanol resulting from standardized fermentation of lactose. The rates of response of the isotopic parameters to changes in the feeding materials is slower for the minor components, proteins, and lipids than for lactose and ethanol. For similar diets, the nonexchangeable sites of lactose and the methyl site of ethanol, in particular, are relatively enriched in deuterium in the case of polygastric animals, cow, goat, and ewe, as compared to the monogastric species, sow and mare, and woman. From an analytical point of view, the carbon and hydrogen parameters of ethanol provide efficient criteria for identifying a whey origin with respect to other agricultural and fossil sources.

Free energy of hydration of a molecular ionic solute: Tetramethylammonium ion

We have performed Monte Carlo simulations of the tetramethylammonium ion, hydrated by 256 simple point charge (SPC) water molecules, as a function of total charge on the ion. The total charge was varied between −3e to +3e at intervals of 1e, and was distributed equally on the four methyl sites on the ion. Derivatives of the free energy with respect to charge were related to the fluctuations in the ion–water interaction energies using a cumulant expansion. This derivative information for the different charge states was found to give an accurate description of the free energy of hydration. The calculated hydration free energies were also found to be only weakly dependent on system size and the method used for calculating the electrostatic interactions (Ewald summation or generalized reaction field), when finite system size corrections are applied. The quadratic charge dependence was obtained for the free energy of hydration for both positive and negative ions as expected from the Born model. The hydration is, however, asymmetric. Negative ions are more favorably hydrated compared to positive ions. We relate this asymmetry of hydration to water structure; that is, to differences in the water oxygen and water hydrogen density profiles surrounding positive and negative ions. Another manifestation of this asymmetry is seen in the positive electrostatic potential at the center of methyl sites in the uncharged state of the tetramethylammonium solute.

Molecular dynamics simulations of dielectric properties of dimethyl sulfoxide: Comparison between available potentials

We present a molecular dynamics simulation study of the orientational correlations of molecular dipoles and dielectric properties of a series of recently proposed models for liquid dimethyl sulfoxide. The dipole–dipole correlations, as measured by the dipolar symmetry projections h110(r) and h112(r), are very similar for the models with four partially charged sites, but differ somewhat for the potential with no charges on the methyl groups. The effects of the charges on the methyl sites upon the local ordering of the molecular dipoles are discussed by comparing the h110(r) projections for two models which differ only in their site-charge assignments. The Kirkwood g factors for the simulated force fields turn out all greater than unity, in disagreement with some experimental estimates. The dielectric constants, however, are in good agreement with reported experimental data. In addition, we discuss the wave vector (k) dependence of the longitudinal static dielectric responses and also the single-particle molecular reorientation dynamics, which is found to differ from model to model. Likely consequences of that to the dielectric relaxation of these models are briefly discussed.

Determination of the potential parameters of a site model from calculations of second virial coefficients of linear and branched alkanes

A previously established method to assign Lennard-Jones parameters for the methyl and methylene site groups in n-alkanes is now generalized to the case of branched alkanes. The method is based on Monte Carlo calculations of the binary cluster integral of the chain molecules, and their comparison with experimental data of second virial coefficients in the gas phase. We have arrived at a single set of values for the parameters epsilon and sigma of the different sites, CH3, CH2, CH and C, that reproduces adequately the virial coefficient data of linear and branched molecules, and also of their mixtures. This way we have also clarified the validity of the different alternative sets proposed in our earlier study for n-alkanes.

Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Fermentation Medium

The isotopic parameters of fermentation products provide reliable criteria for characterizing their carbohydrate precursors on the condition that the isotopic coefficients which relate individual sites in the products and in the reactants have strictly reproducible or predictible values. Since fermentation may be performed either in natural media (such as fruit juices) which exhibit variable compositions or in “synthetic” media prepared from appropriate nutrients, it is necessary to estimate the influence of the medium on the carbon and hydrogen isotope ratios. By combining isotope ratio mass spectrometry and nuclear magnetic resonance investigations it has been shown that variations in the reaction rates induced by the medium are not connected with isotopic fractionation effects. At natural concentrations the nutrients present in juices, for instance, ensure a significant acceleration of the bioconversion with respect to pure water without introducing undesirable isotopic effects. The role of different nutrients added in relatively large amounts to synthetic media has been analyzed. It has been shown in particular that a significant decrease in the isotope ratio of the methylenic site of ethanol is due to the combined influence of both phosphates and assimilable nitrogen which are also the key components governing the growth and activity of yeasts. For a given nutrient composition the effect of the medium on the isotopic coefficients which connect the individual sites of ethanol to the starting water can be estimated. The observed behavior can be explained by modifications in the percentages of intra- and intermolecular hydrogen transfers (methyl site) and by variations in the kinetic isotope effects and exchange rates involving the hydrogens originating from water. Carbohydrates from different origins (beet and cane) have been fermented in different “natural” media prepared by eliminating sugars from orange, apple, and grape juices. In spite of very different fermentation rates the isotopic ratios of the products are then nearly independent of the composition of the medium. These results support the use of the ethanol probe for characterizing endogenous or exogenous sugars contained in different fruit juices.

Natural Abundance Isotopic Fractionation in the Fermentation Reaction: Influence of the Nature of the Yeast

Site-specific natural isotope fractionation studied by nuclear magnetic resonance (SNIF-NMR) provides isotopic criteria that characterize a biochemical transformation such as fermentation and enable isotopic ratios measured in end products to be correlated with those of their precursors. In principle, a given set of transfer coefficients applies only to bioconversions performed under strictly identical conditions, a situation that is hardly fulfilled in most usual fermentation processes. In particular, natural raw materials such as fruits frequently involve complex mixtures of various yeast strains present at different concentrations. Series of experiments performed with different yeasts, different concentrations of car- bohydrates, and different yields of the transformation have shown that, although glycolysis is associated with overall hydrogen fractionation effects that may exceed 40 ppm, the range of variation in the isotopic ratios of the fermentation products, ethanol and water, does not exceed a few parts per million. Provided that the yield in ethanol reaches values higher than 70%, the nature of the yeast strain has minimal influence on the isotopic ratio of the methyl site of ethanol (D/H)I. In contrast, the isotope ratio of the methylene site, (D/H)II, may exhibit significant enhancements, in particular when ethanol is left in contact for a long time with poorly alcohologenic yeasts. These behaviors are consistent with hydrogen transfers from the aqueous medium to the methylene site, and partly to the methyl site, occurring with relatively high kinetic isotope effects. Since water acts as an open pool of hydrogens, however, only small isotopic variations are produced in the course of the fermentation reaction. Moreover, the partial connection between hydrogens from the methyl site of ethanol and hydrogens from glucose operates with relatively small secondary isotopic effects. No significant changes in the percentages of intra- and inter-molecular transfers of hydrogen to the methyl site are observed as a function of the nature of the yeast. These results support the use of the methyl isotopic ratio of ethanol as a probe of the isotopic behavior of carbohydrate precursors, whatever the yeast strains present in natural fermentation media.

Multiple stage pentaquadrupole mass spectrometry for generation and characterization of gas-phase ionic species. The case of the PyC 2H 5+· isomers

Eleven isomers with the PyC2H 5 (+·) composition, which include three conventional (1-3) and eight distonic radical cations (4-11), have been generated and in most cases successfully characterized in the gas phase via tandem-in-space multiple-stage pentaquadrupole MS(2) and MS(3) experiments. The three conventional radical cations, that is, the ionized ethylpyridines C2H5-C5H4N(+·) (1-3), were generated via direct 70-eV electron ionization of the neutrals, whereas sequences of chemical ionization and collision-induced dissociation (CID) or mass-selected ion-molecule reactions were used to generate the distonic ions H2C(·)-C5H4N(+)-CH3 (4-6), CH3-C5H4N(+)-CH 2 (·) (7-9), C5H5N(+)-CH2CH 2 (·) (10), and C5H5N(+)-CH(·)-CH3 (11). Unique features of the low-energy (15-eV) CID and ion-molecule reaction chemistry with the diradical oxygen molecule of the isomers were used for their structural characterization. All the ion-molecule reaction products of a mass-selected ion, each associated with its corresponding CID fragments, were collected in a single three-dimensional mass spectrum. Ab initio calculations at the ROMP2/6-31G(d, p)//6-31G(d, p)+ZPE level of theory were performed to estimate the energetics involved in interconversions within the PyC2H5 (+·) system, which provided theoretical support for facile 4⇌7 interconversion evidenced in both CID and ion-molecule reaction experiments. The ab initio spin densities for the a-distonic ions 4-9 and 11 were found to be largely on the methylene or methyne formal radical sites, which thus ruled out substantial odd-spin derealization throughout the neighboring pyridine ring. However, only 8 and 9 (and 10) react extensively with oxygen by radical coupling, hence high spin densities on the radical site of the distonic ions do not necessarily lead to radical coupling reaction with oxygen. The very typical "spatially separated" ab initio charge and spin densities of 4-11 were used to classify them as distonic ions, whereas 1-3 show, as expected, "localized" electronic structures characteristic of conventional radical ions.

Detection of Added Beet Sugar in Concentrated and Single Strength Fruit Juices by Deuterium Nuclear Magnetic Resonance (SNIF-NMR Method): Collaborative Study

A collaborative study of the site-specific natural isotope fractionation-nuclear magnetic resonance (SNIF-NMR) method for detecting added beet sugar in fruit juices is reported. This method is complementary to the stable carbon isotope ratio analysis (SCIRA) (AOAC Official Methods 981.09 and 982.21), which can detect sugars derived from plants exhibiting C4 metabolism (corn and sugarcane). It is based on the fact that the deuterium content at specific positions of the sugar molecules is higher in fruit sugars than in beet sugar. The fruit juices are fermented, and the alcohol is distilled with a quantitative yield and analyzed with a high-yield NMR spectrometer fitted with a deuterium probe and fluorine lock. The proportion of ethanol molecules monodeuterated on the methyl site is recorded. This parameter (D/H)I is lowered when beet sugar is added to a fruit juice or concentrate. The precision of that method for measuring (D/H)I was observed to be similar to that of other isotope ratio methods: Sr values ranged from 0.19 to 0.25 ppm and SR values varied between 0.21 and 0.37 ppm. An excellent correlation was observed between the percentage of added beet sugar and the (D/H)I isotope ratio measured in this collaborative study. Consequently, all samples in which beet sugar was added were found to have a (D/H)I isotope ratio significantly below the normal value for authentic juice or concentrate of that fruit. The SNIF-NMR method for detection of added beet sugar in fruit juices has been adopted by AOAC INTERNATIONAL.

Spin-Density Distribution, Conformation, and Hydrogen Bonding of the Redox-Active Tyrosine YZ in Photosystem II from Multiple-Electron Magnetic-Resonance Spectroscopies: Implications for Photosynthetic Oxygen Evolution

The oxidized form of the redox-active Y{sub Z} tyrosyl residue involved in photosynthetic oxygen evolution has been generated and trapped in Mn-depleted Photosystem II core complexes from a D2-Y160F mutant strain of Synechocystis 6803. This system eliminates interference from P{sub 700}{sup +} and Y{sub D}D+{center_dot} and allowed characterization of Y{sub Z}{sup {center_dot}} by using a combination of specific {sup 2}H-labeling and electron magnetic-resonance techniques that included CW-EPR, frequency-modulated and transient detected ENDOR, and {sup 2}H-ESEEM. Using these complementary techniques, we have carried out a detailed evaluation of the hyperfine structure of Y{sub Z}{sup {center_dot}} and obtained the dipolar interactions to weakly coupled nuclei, the strongly anisotropic tensors of the ring-hydrogens, and the more isotropic interactions to the {Beta}-methylene site. We conclude that tyrosyl radicals are not tuned to specific function by large-scale modulations of their spin density through hydrogen-bonding effects. We suggest a hydrogen-atom transfer function for Y{sub Z} in water oxidation. Within this model, the (Mn){sub 4}/Y{sub Z} center forms the Oxygen-Evolving Complex of Photosystem II where the (Mn){sub 4} cluster binds substrate water and delocalizes oxidizing equivalents and Y{sub Z} acts by abstracting hydrogens from substrate water in either a concerted or sequential fashion. 71 refs., 8 figs.,more » 2 tabs.« less

Self-Assembled .omega.-Hydroxyalkanethiol Monolayers with Internal Functionalities: Electrochemical and Infrared Structural Characterizations of Ether-Containing Monolayers

The structure of w-hydroxyalkanethiol monolayers containing an intemal ether group is probed by traditional surface infrared reflection-absorption spectroscopy and a novel electrochemical method based on the measured shifts in the potential of zero charge (pzc) upon introduction of the ether group. The analyses of the infrared and electrochemical data give complementary structural information. Vibrational bandwidth measurements indicate that these monolayers exhibit crystalline packing. From a comparison of the integrated infrared absorption intensities between monolayer and bulk samples of the thiols, an average tilt angle of 29 and an average twist angle of are determined for the ether modified w-hydroxyalkanethiol monolayers. The pzc of the ether modified monolayer coated Au electrodes is observed to shift by 130 i 40 mV from that characteristic of an unmodified w-hydroxyalkanethiol monolayer. A simple dipole analysis of the magnitude of these pzc shifts, assuming a twist angle of 40, gives an average tilt angle of 23 & 8, in reasonable agreement with the infrared determination. In addition, the monolayers modified by an oxygen located at an even methylene site (from the S atom) are observed to give a positive pzc shift, while those located at odd sites give strictly negative pzc shifts. Such an evedodd effect on the sign of the pzc shift indicates that the S-C bond is directed away from the surface normal. This electrochemical approach to structural analysis is generally applicable to crystalline monolayers which contain a net static internal dipole.

NMR investigation of the intramolecular distribution of deuterium in natural triacylglycerols

AbstractThe 1H and 2H NMR spectra of a number of edible oils were investigated. The average numbers of hydrogens and deuteriums for the olefinic, methylene and methyl sites in mixtures of triacylglycerols were determined, and some parameters describing the different fractionation of deuterium in the various sites were obtained. These results are discussed in connection with the biosynthesis steps involved in the elongation and desaturation processes of the fatty acid components.

Dynamics and structure of a flexible columnar liquid crystal based on tetrabenzocyclododecatetraene

Abstract The dynamical and conformational behaviour of a flexible tetrabenzocyclododecatetraene derivative exhibiting a columnar mesophase has been studied by a combination of deuteron solid state NMR spectroscopy and molecular dynamics (MD) simulations. As shown by two-dimensional (2D) exchange NMR, the mesophase is characterized by slow axial reorientations (∼10−3s) of single molecular units where the phenylene rings exhibit a well-defined quasi-fourfold potential, while the 2D spectra of the core methylene sites are sensitive to the molecular conformation and reorientation mechanism. Motional narrowing of one-dimensional (1D) spectra reveals additional fast librations due to the internal flexibility of the mesogenic moiety. The various reorientation pathways comprising interconversions and pseudo-rotations between different energetically stable conformations are elucidated on a microscopic level by molecular dynamics simulations. The mesophase dynamics is ascribed to a complex axial motion involving ro...

Thermal and mechanical characterization of poly[bis(4-benzylphenoxy)phosphazene

In this paper we report the thermal and mechanical characterization of poly[bis(4-benzylphenoxy)phosphazene] (PBPP). Glass transition and melting temperatures of 13.8 and 88.2°C respectively, and heat of fusion of 7.0 J g −1 were measured by differential scanning calorimetry (d.s.c.) for the as-synthesized polymer. Crystallization studies from the melt, in both isothermal and dynamic regimes, showed a low crystallizability of PBPP, due to the high mobility and steric hindrance of lateral substituents, with a heat of fusion no higher than 9 J g −1. Activation energies of about 70 kJ mol −1 were calculated both for the crystallization process and for the rubber-glass transition in the amorphous state. The highest crystallization rate in isothermal experiments occurred at a temperature of 45°C. Thermodegradation of PBPP in air was found to start with the probable formation of the hydroperoxide group on the methylene site at about 233°C, as demonstrated from both thermogravimetric and d.s.c. analyses. PBPP film behaved as an elastomeric polymer with a strain at break higher than 350%. A tensile modulus of 1.2 MPa and a stress at break of 4.8 MPa for the amorphous polymer were measured, whereas crystalline polymers exhibited higher tensile modulus and stress at break, ranging from 34 to 48 MPa and from 7 to 12 MPa, respectively.

Interregional and interannual variability of ethanol site-specific deuterium content in Italian wines

The site-specific natural isotope fractionationnuclear magnetic resonance (SNIF-NMR)2H analysis is at present the reference method for determining the origin of ethanol and has been successfully applied for analysing all grape products. The method has also been used to identify the origin of wines and the aim of this study was to verify this application. In all, 3309 samples belonging to the Italian wines data bank were analysed from all 20 Italian regions to study the possibilities of determining and controlling the exact origin and year of the product. The dependency of the deuterium/hydrogen ratio of the methyl site (D/HI) with latitude was confirmed but this was not the case for the D/H ratio of the methylene site (D/HII) and the parameter R [2(D/HII)/(D/HI)] did not add any additional information. The frequency distribution of the NMR parameters follows a regular North-South gradient but they are not sufficiently different in each region to allow unique characterisation. The lack of effectiveness of the method, when applied to identification, depends on interannual and interregional variability. As a consequence, all but some very different regions overlap. The SNIF-NMR method is particularly suitable for detecting simple adulterations but, in the Italian situation, seems not to be so useful for solving problems of geographical origin. For this, it could be helpful to use other isotopic parameters and to ameliorate the sampling technique, to improve the effectiveness of the method.

Reactions of tert-butoxyl radicals with acyclic ethers studied by the radical trapping technique

1,1,3,3-Tetramethyl-2,3-dihydro-1H-isoindol-2-yloxyl has been used as a radical trap to investigate the pattern of hydrogen abstraction reactions occurring in a range of acyclic ethers by tert-butoxyl radicals. The results confirm the high degree of selectivity for abstraction at C atoms adjacent to ethereal O. The presence of two α ethereal O atoms is less effective in enhancing abstraction than one. This is possibly due to a stereoelectronic effect whereby the 1,3-oxygen orbital interactions reduce the ability of either oxygen to interact effectively with the developing radical centre. Whereas the presence of an α-oxygen enhances the rate of hydrogen abstraction, the presence of a β-oxygen retards the rate of hydrogen abstraction. Abstraction at a methine C atom adjacent to ethereal O occurs at about the same rate or a little faster than at a methylene C atom, whereas abstraction at methyl groups is much slower. A temperature study of the abstraction from methyl and methylene sites in dimethoxymethane shows that the difference in abstraction rates at these sites is predominantly an entropy effect. A lower entropy of activation due to the loss of the internal rotational mode of the methyl group in the formation of the transition state is the probable reason. Ethers appear to be less reactive than alcohols in hydrogen abstraction reactions.

Measurement of two- and three-bond 13C?1H J couplings to the C? carbons of leucine residues in staphylococcal nuclease

A new 1H-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond 13C-1H couplings in proteins with selectively 13C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 13C]-labeled in all 11 leucine positions and ligated with thymidine 3',5'-biphosphate and Ca2+. Two- and three-bond 13C methyl-proton couplings are reported and, together with the measured three-bond JC alpha C delta in uniformly 13C-enriched staphylococcal nuclease, the chi 2-angles and the stereospecific assignments of the C delta methyl group with respect to the prochiral beta-protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of 13C relaxation times have measured coupling constants that are indicative of motional averaging.

Uracil-DNA glycosylase as a probe for protein--DNA interactions.

The DNA repair enzyme Uracil-DNA Glycosylase (UDG) can be used to investigate three different features of protein-DNA interactions. Complexes can be probed by simple protection experiments ('footprinting') or by two kinds of interference assays: a missing thymine site (MT-site) experiment and a missing thymine methyl site (MTM-site) experiment. The three probing methods are assessed using the well-characterized in vitro systems of lambda repressor and lac repressor binding to their respective operator sites. The results obtained with UDG probing agree well with previous probing experiments on the same systems and, in certain cases, extend previous interpretations: for example, comparison of the results obtained with the two interference assays shows that formation of the lac repressor-operator complex requires interactions with the methyl group of one particular thymine residue (T-13) in the operator but also requires interactions with other parts of the thymine base at operator positions 7, 8, 9, 21, 23 and 24. Overall, the properties of UDG recommend it as a versatile and convenient method to investigate DNA-protein interactions both in vitro and possibly in vivo.

Intramolecular photoreaction of synthetic oligopeptide-linked anthraquinone molecules

Photoreaction of (N-acetylglycyl)oligopeptide-linked anthraquinone molecules was investigated. In an acetonitrile solution, the photoexcited anthraquinone moiety abstracted intramolecularly the hydrogen atom of the methylene site of glycine residue. The biradical formed was followed by the formation of C-O bonding via radical recombination to produce ring-closure products in high yields (23-58%). A variety of oligopeptide spacers between acetylglycine and anthraquinone moieties were systematically changed, and their photoreactivities were investigated

Nonlinear least‐squares fitting procedure for solid‐state NMR powder patterns

AbstractA fitting procedure for the extraction of the characteristic NMR parameters from solid‐state NMR powder patterns is described at an introductory level. The Levenberg‐Marquardt nonlinear least‐squares algorithm is recoded in Matlab, a vector‐oriented programming language, so that the NMR simulation programs are more condensed than are those in traditional programming languages. Three examples of line‐shape fitting are shown: the deuterium powder pattern for the bridging deuterium site in [Et4N] [2HCr2(CO)10], the proton‐coupled 13C powder pattern for the bridging methylene site in cis‐(μ‐CH2)(μ‐CO)[FeCp(CO)]2, and the (14N, 195Pt)‐coupled 13C powder pattern for the Pt‐CN system in K2Pt(CN)4 · 3H2O. The results provide information about quadrupolar, dipolar, and chemical‐shielding tensors. In each case, the line‐shape is calculated from the spin Hamiltonian expressed in a simple, uncoupled basis set. The variables in each case are a small set of NMR parameters: the quadruple‐coupling constant and the asymmetry parameter. The effects of finite pulse‐lengths or anisotropic cross‐polarization are not included in the line‐shape simulations. The line‐shapes are powder patterns; averaging over the Euler angles, θ and x, is done either with constant angle step size or by the Conroy‐Wolfsberg tiling scheme.