Chemical Shift Measurements Research Articles

PISEMA Solid‐State NMR Spectroscopy

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Chemical Effects in High-Resolution Nickel Kα X-Ray Fluorescence Spectra

The nickel Kalpha spectra of oxides, halides (NiF2, K2NiF6, NiCl2, NiBr2), complex compounds, and metal are measured with two different double-crystal X-ray fluorescence spectrometers. The peak shifts and line width changes due to the changes in the chemical states are reported. High reproducibility has been shown for the chemical shift and line width measurements. The DV-Xalpha molecular orbital calculation at the ground and the 1s(-1) hole states was performed to prove that the chemical effect originats from the charge-transfer mechanism.

NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain.

The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A 1H–15N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a 15N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA701–168) and p53TAD. HRPA701–168 residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD–hRPA70 complex. The amide 1H and 15N chemical shifts of an uniformly 15N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA701–168. In the presence of unlabeled hRPA701–168, resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42–56. Minimal binding was observed between p53TAD and a mutant form of hRPA701–168, where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA701–168 was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA701–168. 15N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA701–168. Large changes in both the transverse (R2) and rotating frame (R1ρ) relaxation rates were observed for a subset of the p53TAD residues that had 1H–15N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R2 and R1ρ. A model that couples the formation of a weak encounter complex between p53TAD and hRPA701–168 to the folding of p53TAD is discussed in the context of a functional role for the p53–hRPA70 complex in DNA repair.

Hyperpolarized 129Xe NMR investigation of multifunctional organic/inorganic hybrid mesoporous silica materials

An extensive study has been made on a series of multifunctional mesoporous silica materials, prepared by introducing two different organoalkoxysilanes, namely 3-[2-(2-aminoethylamino)ethylamino]propyltrimethoxysilane (AEPTMS) and 3-cyanopropyltriethoxysilane (CPTES) during the base-catalyzed condensation of tetraethoxysilane (TEOS), using the variable-temperature (VT) hyperpolarized (HP) 129Xe NMR technique. VT HP-129Xe NMR chemical shift measurements of adsorbed xenon revealed that surface properties as well as functionality of these AEP/CP-functionalized microparticles (MP) could be controlled by varying the AEPTMS/CPTES ratio in the starting solution during synthesis. Additional chemical shift contribution due to Xe-moiety interactions was observed for monofunctional AEP-MP and CP-MP as well as for bifunctional AEP/CP-MP samples. In particular, unlike CP-MP that has a shorter organic backbone on the silica surface, the amino groups in the AEP chain tends to interact with the silanol groups on the silica surface causing backbone bending and hence formation of secondary pores in AEP-MP, as indicated by additional shoulder peak at lower field in the room-temperature 129Xe NMR spectrum. The exchange processes of xenon in different adsorption regions were also verified by 2D EXSY HP-129Xe NMR spectroscopy. It is also found that subsequent removal of functional moieties by calcination treatment tends to result in a more severe surface roughness on the pore walls in bifunctional samples compared to monofunctional ones. The effect of hydrophobicity/hydrophilicity of the organoalkoxysilanes on the formation, pore structure and surface property of these functionalized mesoporous silica materials are also discussed.

Structural and Dynamic Independence of Isopeptide-linked RanGAP1 and SUMO-1

Although sumoylation regulates a diverse and growing number of recognized biological processes, the molecular mechanisms by which the covalent attachment of the ubiquitin-like protein SUMO can alter the properties of a target protein remain to be established. To address this question, we have used NMR spectroscopy to characterize the complex of mature SUMO-1 with the C-terminal domain of human RanGAP1. Based on amide chemical shift and 15N relaxation measurements, we show that the C terminus of SUMO-1 and the loop containing the consensus sumoylation site in RanGAP1 are both conformationally flexible. Furthermore, the overall structure and backbone dynamics of each protein remain unchanged upon the covalent linkage of Lys524 in RanGAP1 to the C-terminal Gly97 of SUMO-1. Therefore, SUMO-1 and RanGAP1 behave as "beads-on-a-string," connected by a flexible isopeptide tether. Accordingly, the sumoylation-dependent interaction of RanGAP1 with the nucleoporin RanBP2 may arise through the bipartite recognition of both RanGAP1 and SUMO-1 rather than through a new binding surface induced in either individual protein upon their covalent linkage. We hypothesize that this conformational flexibility may be a general feature contributing to the recognition of ubiquitin-like modified proteins by their downstream effector machineries.

Local Aromaticity of the Six-Membered Rings in Pyracylene. A Difficult Case for the NICS Indicator of Aromaticity

In this work, we have analyzed the local aromaticity of the six-membered rings (6-MRs) of planar and pyramidalized pyracylene species through the structurally based harmonic oscillator model of aromaticity (HOMA), the electronically based para-delocalization index (PDI), and the magnetic-based nucleus independent chemical shift (NICS) measurements, as well as with maps of ring current density. According to ring currents and PDI and HOMA indicators of aromaticity, there is a small reduction of local aromaticity in the 6-MRs of pyracylene with a bending of the molecule. In the case of NICS, the results depend on whether the NICS value is calculated at the center of the ring (NICS(0)) or at 1 A above (NICS(1)(out)) or below (NICS(1)(in)) the ring plane. While NICS(1)(out) values also indicate a slight decrease of aromaticity with bending, NICS(0) and NICS(1)(in) wrongly point out a large increase of aromaticity upon distortion. We have demonstrated that the NICS(0) reduction in the 6-MRs of pyracylene upon bending is due to (a) a strong reduction of the paratropic currents in 5-MRs and (b) the fact that, due to the distortion, the paratropic currents point their effects in other directions.

NMR crystallography: the use of chemical shifts

Measurements of chemical shifts obtained from magic-angle spinning NMR spectra (together with quantum mechanical computations of shielding) can provide valuable information on crystallography. Examples are given of the determination of crystallographic asymmetric units, of molecular symmetry in the solid-state environment, and of crystallographic space group assignment. Measurements of full tensor components for 199Hg have given additional coordination information. The nature of intermolecular hydrogen bonding in cortisone acetate polymorphs and solvates is obtained from chemical shift information, also involving measurement of the full tensor parameters. The resulting data have been used as restraints, built into the computation algorithm, in the analysis of powder diffraction patterns to give full crystal structures. A combination of quantum mechanical computation of shielding and measurement of proton chemical shifts (obtained by high-speed MAS) leads to the determination of the position of a proton in an intermolecular hydrogen bond. A recently-developed computer program specifically based on crystallographic repetition has been shown to give acceptable results. Moreover, NMR chemical shifts can distinguish between static and dynamic disorder in crystalline materials and can be used to determine modes and rates of molecular exchange motion.

Tilt and rotational pitch angle of membrane-inserted polypeptides from combined 15N and 2H solid-state NMR spectroscopy.

Knowledge of the alignment of alpha-helical polypeptides with respect to the membrane surface and their dynamics in the membrane are key to understanding the functional mechanisms of channels, antibiotics, and signal or translocation peptides. In this paper polypeptides have been labeled with [3,3,3-(2)H(3)]alanine as well as with (15)N at single site amide positions and reconstituted into oriented phospholipid bilayers. A transmembrane and two amphipathic helical polypeptides with the deuterium label at orthogonal positions have been investigated by deuterium and proton-decoupled (15)N solid-state NMR spectroscopy. The (15)N chemical shift measurements and the deuterium quadrupole splitting exhibit a highly complementary functional dependence with respect to the spatial alignment of the polypeptide. Therefore, the combination of these two measurements allows one to determine both the tilt and the rotational pitch angle with high precision. In addition, the deuterium line shape is very sensitive to mosaic spread and the relative orientation of the peptide. The solid-state NMR measurements indicate that the model sequences exhibit a small degree of mosaicity, when at the same time the phospholipid headgroup region is significantly distorted. Furthermore, the (2)H solid-state NMR spectra reveal small orientational and dynamic differences when the fatty acyl chain composition of the phosphatidylcholine bilayers is modified.

G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment

A G-matrix Fourier transform (GFT) NMR spectroscopy-based strategy for resonance assignment of proteins is described. Each of the GFT NMR experiments presented here rapidly affords four-, five-, or six-dimensional spectral information in combination with precise measurements of chemical shifts. The resulting high information content enables one to obtain nearly complete assignments by using only four NMR experiments. For the backbone amide proton detected "out-and-back" experiments, data collection was further accelerated up to approximately 2.5-fold by use of longitudinal (1)H relaxation optimization. The GFT NMR experiments were acquired for three proteins with molecular masses ranging from 8.6 to 17 kDa, demonstrating that the proposed strategy is of key interest for automated resonance assignment in structural genomics.

NMR studies of the inclusion complex between beta-cyclodextrin and paroxetine.

A 1H and 13C NMR study on the inclusion complex of paroxetine with beta-cyclodextrin was carried out in order to define the stoichiometry of the association and its strength. Proton and carbon chemical shift measurements of paroxetine and beta-cyclodextrin were performed at several molar ratios and temperatures, allowing the determination of a 1:1 stoichiometry and an association constant value of the order of 2 x 10(3) for the paroxetine-beta-cyclodextrin complex. Overhauser effects in the rotating frame were also measured, and the experimental interproton distance constraints have been used for molecular model building of the complex. The obtained model indicates that the benzodioxolyl moiety of paroxetine is deeply inserted in the cavity of the cylindrical structure of beta-cyclodextrin, while the fluoro-phenyl ring lays above the wider rim.

Correlation between chemical shift of SiKαlines and the effective charge on the Si atom and its application in the Fe-Si binary system

High-resolution Si $K{\ensuremath{\alpha}}_{1,2}{(K\ensuremath{-}L}_{3,2})$ x-ray fluorescence spectra of Si, ${\mathrm{SiB}}_{6},$ SiC, ${\mathrm{Si}}_{3}{\mathrm{N}}_{4},{\mathrm{SiO}}_{2},$ and ${\mathrm{Na}}_{2}{\mathrm{SiF}}_{6}$ were measured on a double-crystal spectrometer. From ${\mathrm{SiB}}_{6}$ to ${\mathrm{Na}}_{2}{\mathrm{SiF}}_{6},$ the chemical shifts for the Si $K\ensuremath{\alpha}$ lines relative to pure Si were 0.05, 0.19, 0.45, 0.62, and 0.96 eV, respectively. The value increased systematically with increased atomic number of the neighbor atoms of Si. Calculation by the discrete variational Hartree-Fock-Slater method showed that the effective charges on Si in SiC and ${\mathrm{Na}}_{2}{\mathrm{SiF}}_{6}$ were 0.37 and 1.85 electrons, and the values in ${\mathrm{Si}}_{3}{\mathrm{N}}_{4},{\mathrm{SiO}}_{2}$ were found in former literature to be 1.24 and 1.40 electrons, which increased in the same sequence as the chemical shift. These facts certified the direct correlation between the effective charges on Si and the chemical shift of Si $K\ensuremath{\alpha}$ lines. An empirical curve of effective charges on Si versus chemical shifts in Si $K\ensuremath{\alpha}$ lines was obtained. With this empirical curve, we found that Si held positive charge in the Fe-Si binary system based on the Si $K\ensuremath{\alpha}$ chemical shift measurement, although the electronegativity of Si (1.90) was greater than that of Fe (1.83), which made it seem reasonable to assume that the charge transfer should be from Fe to Si. We determined the effective charges on Si in ${\mathrm{FeSi}}_{2},$ FeSi, and Si steel to be 0.50, 0.56, and 0.63 electrons, which increased with the increasing Fe concentration.

Temperature dependence and resonance assignment of 13C NMR spectra of selectively and uniformly labeled fusion peptides associated with membranes.

HIV-1 and influenza viral fusion peptides are biologically relevant model fusion systems and, in this study, their membrane-associated structures were probed by solid-state NMR (13)C chemical shift measurements. The influenza peptide IFP-L2CF3N contained a (13)C carbonyl label at Leu-2 and a (15)N label at Phe-3 while the HIV-1 peptide HFP-UF8L9G10 was uniformly (13)C and (15)N labeled at Phe-8, Leu-9 and Gly-10. The membrane composition of the IFP-L2CF3N sample was POPC-POPG (4:1) and the membrane composition of the HFP-UF8L9G10 sample was a mixture of lipids and cholesterol which approximately reflects the lipid headgroup and cholesterol composition of host cells of the HIV-1 virus. In one-dimensional magic angle spinning spectra, labeled backbone (13)C were selectively observed using a REDOR filter of the (13)C-(15)N dipolar coupling. Backbone chemical shifts were very similar at -50 and 20 degrees C, which suggests that low temperature does not appreciably change the peptide structure. Relative to -50 degrees C, the 20 degrees C spectra had narrower signals with lower integrated intensity, which is consistent with greater motion at the higher temperature. The Leu-2 chemical shift in the IFP-L2CF3N sample correlates with a helical structure at this residue and is consistent with detection of helical structure by other biophysical techniques. Two-dimensional (13)C-(13)C correlation spectra were obtained for the HFP-UF8L9G10 sample and were used to assign the chemical shifts of all of the (13)C labels in the peptide. Secondary shift analysis was consistent with a beta-strand structure over these three residues. The high signal-to-noise ratio of the 2D spectra suggests that membrane-associated fusion peptides with longer sequences of labeled amino acids can also be assigned with 2D and 3D methods.

Aggregation Behavior of Fluorooctanols in Hydrocarbon Solvents.

The association behaviors of three 1-octanols (1-octanol: C8OH; 1,1,2,2-tetrahydrotridecafluorooctanol: TFC8OH; and 1,1-dihydropentadecafluorooctanol: DFC8OH) in two hydrocarbon solvents (n-hexane and benzene) were examined by vibration spectroscopy from 288.15 to 318.15 K. From the analysis of results with a mass action model, it was found that dimers and tetramers of 1-octanols coexisted with monomers in the n-hexane solution. These aggregates were formed by hydrogen bonding between the OH groups of 1-octanols. In the n-hexane solutions, an increase in the fluorination number of the 1-octanol molecule enhanced the intermolecular hydrogen bonding between the OH groups, but reduced the amounts of polymeric species. Conversely, in the benzene solution, the NIR experiment suggested that the OH groups of 1-octanols did not interact with other OH groups, but with the benzene molecules instead. It was found from (19)F NMR chemical shift measurements that the fluorooctanols in the benzene solution aggregated by interaction between the fluorocarbon chains instead of by hydrogen bonding.

Atomic refinement with correlated solid-state NMR restraints

The orientation data provided by solid-state NMR can provide a great deal of structural information about membrane proteins. The quality of the information provided is, however, somewhat degraded by sign degeneracies in measurements of the dipolar coupling tensor. This is reflected in the dipolar coupling penalty function used in atomic refinement, which is less capable of properly restraining atoms when dipolar sign degeneracies are present. In this report we generate simulated solid-state NMR data using a variety of procedures, including back-calculation from crystal structures of α-helical and β-sheet membrane proteins. We demonstrate that a large fraction of the dipolar sign degeneracies are resolved if anisotropic dipolar coupling measurements are correlated with anisotropic chemical shift measurements, and that all sign degeneracies can be resolved if three data types are correlated. The advantages of correlating data are demonstrated with atomic refinement of two test membrane proteins. When refinement is performed using correlated dipolar couplings and chemical shifts, perturbed structures converge to conformations with a larger fraction of correct dipolar signs than when data are uncorrelated. In addition, the final structures are closer to the original unperturbed structures when correlated data are used in the refinement. Thus, refinement with correlated data leads to improved atomic structures. The software used to correlate dipolar coupling and chemical shift data and to set up energy functions and their derivatives for refinement, CNS-SS02, is available at our web site.

Theory, simulation and measurement of chemical mass shifts in RF quadrupole ion traps

A comprehensive description of chemical mass shifts in the RF quadrupole ion trap (Paul trap) is given and their origin is explained. Extending a previously proposed model, it is shown that chemical mass shifts are a result of an ejection delay caused by field imperfections in RF ion traps with non-optimized geometry, in particular, field imperfections resulting from holes in the end-cap electrodes, and of compound-dependent modifications of this ejection delay by collisions of the ions with the buffer gas. Elastic collisions change peak shapes and peak widths, but give rise to only very small chemical mass shifts. Inelastic collisions, which result in dissociation, can lead to large chemical mass shifts. The field imperfections present in real ion traps are examined, and differences in the ion ejection behavior and in the chemical mass shifts occurring in boundary ejection and resonance ejection scans are investigated. The dependence of the shifts on the trap geometry, on the buffer gas pressure, on the RF amplitude scan rate, on ion mass, and on ion chemical structure, are explained. The proposed model for chemical mass shifts is validated through experimental measurements and quantitative simulations using the ion trap simulation program ITSIM. It is shown that peak shapes in mass spectra can be reproduced or predicted by simulations, for ion traps with a variety of geometries and operating conditions. Very good agreement between simulations and experiments is found, provided experimental and simulated geometries differ slightly in the end-cap separation, indicating that additional previously neglected field imperfections may be present in common RF ion traps.

The inside story of fullerene anions: a 3He NMR aromaticity probe.

Fullerene anions with helium atoms contained inside the cage exhibit degrees of aromaticity that can be predicted through the measurement of 3He chemical shifts (see picture). These chemical shifts help to demonstrate that the magnetic properties of fullerenes, and their anions, are not simply related to the number of carbon atoms or the number of electrons in the π system.

Structural and dynamical changes of the bindin B18 peptide upon binding to lipid membranes. A solid-state NMR study.

Structural and dynamical features of the B18 peptide from the sea urchin sperm binding protein were determined in the crystalline state and in zwitterionic lipid bilayers at a peptide:lipid molar ratio of 1:12 using solid-state NMR spectroscopy. The study was focused on three (13)C and (15)N uniformly labeled leucine residues, which were introduced into three different B18 peptides at positions evenly distributed along the B18 primary structure. Isotropic (13)C and (15)N chemical shift measurements showed that while B18 possesses a nonhelical and non-sheet-like structure in the crystalline state, the peptide adopts an oligomeric beta-sheet structure in the membrane in the presence of Zn(2+) ions at high peptide:lipid ratio. Torsion angle measurements for the three leucine sites supported these results, with phi torsion angles between -80 degrees and -90 degrees in the crystalline state and between -110 degrees and -120 degrees in the membrane-bound form. These phi torsion angles determined for membrane-bound B18 are consistent with a parallel beta-sheet secondary structure. Analysis of motionally averaged dipolar coupling measurements established an increase of the mobility in the leucine side chains upon binding to the membrane, whereas the backbone mobility remained essentially unchanged, except in the binding site of Zn(2+) ions. This difference in mobility was related to the H-bond network in the parallel beta-sheet structure, which involves the backbone and excludes the side chains of leucine residues. The parallel beta-sheet structure of B18 in the membrane in the presence of Zn(2+) appears to be an active state for the fusion of zwitterionic membranes in the presence of Zn(2+). A fluorescence fusion assay indicated that high B18 concentrations are required to induce fusion in these systems. Therefore, it was hypothesized that the oligomeric beta-sheet secondary structure revealed in the study represents an active state of the peptide in a membrane environment during fusion.

Improved quantitative solution state 13C NMR analysis of ethylene-1-octene copolymers

In order to acquire fully quantitative, high signal-to-noise 13C NMR spectra of ethylene-1-octene copolymers in a relatively short period of time, a detailed protocol has been developed, based on the addition of an optimised amount of the relaxation agent chromium(III)triacetylacetonate and without using nuclear overhauser enhancement (NOE). Compared to classical measurements with NOE, the proposed measuring protocol additionally offers a gain in signal-to-noise of 16%, which corresponds to a gain in experimental time of 34%. It allows precise and accurate quantification of all resonances, including small one's related to, e.g. comonomer inversion, and offers more refined information towards the determination of chemical shift and peak area measurements and the development of statistical chain microstructure models.

NMR Studies of Equilibrium Quotient of the Benzonitrile with Xylene Isomers and Ethylbenzene

The charge transfer (CT) complex formation of benzonitrile as acceptor with the aromatic donors o-, m-, p-xylene and ethylbenzene in CCl4 solutions is investigated by chemical shift measurements relative to an external reference. The equilibrium parameters, Q and DAD of their weak molecular complexes, as found numerically modified Cresswel- Allred (C-A) method, is compared with graphically Scatchard-Foster-Fyfe (Sc-F-F) method.

NMR Measurement of Identical Polymer Samples by Round Robin Method V. Determination of Degree of Polymerization for Isotactic Poly(methyl methacrylate) Having a t-Butyl End Group

Research Group on NMR, The Society of Polymer Science, Japan, has continued assessments of the reliability of NMR measurements of polymers. In the present study, 1H and 13C NMR data for isotactic poly(methyl methacrylate) (it-PMMA) having a t-butyl end group were collected on 27 NMR spectrometers by a round-robin method, to survey the accuracy of NMR spectroscopic determination of degree of polymerization (DP) of the polymer by the end group analysis as well as other basic NMR parameters such as chemical shift and spectral resolution. Five samples of it-PMMAs whose DP’s range from 53 to 5200 were prepared with t-BuMgBr in toluene. 1H and 13C NMR spectra were measured in nitrobenzene-d5 at 110 °C. The standard deviations (σ) for chemical shift measurements of 1H and 13C NMR signals were 0.001–0.004 ppm and 0.04–0.18 ppm, respectively. A singlet signal due to t-butyl group at initiating chain end of the it-PMMA is separately observed from the signals due to the monomeric units in 1H NMR spectra measured in nitrobenzene-d5. Thus DP of the polymer can be determined by the equation of DP = 3 × [OCH3]/[t-C4H9]. The value of σ for DP determination by 1H NMR increased as DP increased (6.4–23.4%). However, the averaged values of DP agreed well with those determined by SEC. 13C NMR analysis of DP was possible for the it-PMMA with DP up to ca. 250. The values agreed with those determined by 1H NMR spectroscopy. 1H NMR determination of DP by 5 runs using single spectrometers (100 and 500 MHz) gave much better reproducibility; σ 3.9–9.2% and 0.7–3.4% for the measurements with 100 and 500 MHz instruments, respectively.