High-speed Magic-angle Spinning Research Articles

Characterization of nucleosome sediments for protein interaction studies by solid-state NMR spectroscopy.

Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ~ 200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation. Since nucleosomes are known to self-associate, these dense phases may induce extensive interactions between nucleosomes, which could interfere with protein-binding studies. Here, we characterized the packing of nucleosomes in the dense phase created by sedimentation using NMR and small-angle X-ray scattering (SAXS) experiments. We found that nucleosome sediments are gels with variable degrees of solidity, have nucleosome concentration close to that found in crystals, and are stable for weeks under high-speed magic angle spinning (MAS). Furthermore, SAXS data recorded on recovered sediments indicate that there is no pronounced long-range ordering of nucleosomes in the sediment. Finally, we show that the sedimentation approach can also be used to study low-affinity protein interactions with the nucleosome. Together, our results give new insights into the sample characteristics of nucleosome sediments for ssNMR studies and illustrate the broad applicability of sedimentation-based NMR studies.

Fast Magic-Angle Spinning Solid-State 1H NMR Reveals Structural Relationships in the High Explosive 2,6-Diamino-3,5-dinitropyrazine-1-oxide (LLM-105)

1H nuclear magnetic resonance (NMR) experiments were performed on the proposed insensitive high-explosive 2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105) at high-speed magic-angle spinning rates of up to 60 kHz. These rapid speeds produce well-resolved spectra that aid in the study of the chemical and structural properties of this material. Extraordinarily long 1H T1 values were observed for the main amine peaks from the LLM-105 explosive and can be used to differentiate between peaks from the main compound and those of impurities. Further, advanced NMR measurements and simulations reveal unique spectral properties due to the strong network of intramolecular and intermolecular hydrogen bonding and provide insights in the structure of LLM-105.

High-Resolution Solid-State NMR Investigation of the Phase Transition in Decamethylferrocene−Acenaphthenequinone Charge-Transfer Complex

A charge-transfer complex composed of decamethylferrocene (D) and acenaphthenequinone (A) was prepared. The material was a 1:1 neutral complex with a mixed-stack structure and exhibited a phase transition at -16 degrees C. High-resolution (13)C and (1)H NMR spectroscopy revealed that an inclination of A with respect to D occurs below the phase-transition temperature. The (1)H spin-diffusion rates of the complex undergoing high-speed magic-angle spinning (MAS) were measured to determine the shortest (1)H-(1)H distance r between D and A. To analyze the experimental results, we derived the analytical expression of the spin-diffusion rate W(z) for a homonuclear multispin system undergoing MAS. It was found that W(z) for the complex is proportional only to 1/r(6) under high-speed MAS conditions. On the basis of this relationship and the crystal structure at 20 degrees C, it was determined that the shortest (1)H-(1)H distance r at -27.7 degrees C (below the phase transition temperature) is 0.4 A shorter than that at 20 degrees C. Given this information, a plausible model of the low-temperature structure is discussed.

Crosslinking junctions of vulcanized natural rubber analyzed by solid-state NMR spectroscopy equipped with field-gradient-magic angle spinning probe

Crosslinking junctions of vulcanized natural rubber were analyzed by solid-state NMR spectroscopy equipped with a field-gradient high speed magic angle spinning probe. Resolution of 1H and 13C NMR spectra and correlations between 1H and 13C of the vulcanized natural rubber were investigated by one- and two-dimensional measurements, including inverse correlation measurements. The number of substitution of the carbon atom at the crosslinking junctions was determined by solid-state NMR spectroscopy.

1H homonuclear dipolar decoupling at high magic-angle spinning frequencies with rotor-synchronised symmetry sequences

We demonstrate here application of symmetry-adapted sequences of the form RN n ν to obtain high-resolution 1H spectra in solid-state nuclear magnetic resonance under high-speed magic-angle spinning. Experimental results are shown for samples of alanine and glycine for spinning speeds up to 30 kHz with radio-frequency nutation rates of around 100 kHz.

27Al NMR Study of the Structure of Lanthanum- and Yttrium-Based Aluminosilicate Glasses and Melts

We have investigated by (27)Al nuclear magnetic resonance spectroscopy some compositions in the Ln(2)O(3)-Al(2)O(3)-SiO2 (Ln = Y or La) ternary phase diagram containing more than 60 mol % of SiO2. One- and two-dimensional high-field (17.6 T) high-speed (30 kHz) magic angle spinning experiments have been performed along with simulations of the spectra to quantify the amount of penta-coordinated aluminum present in those glasses as a function of composition. Very high-temperature experiments have allowed to follow selected samples from 2200 degrees C down to 1700 degrees C and hence to characterize the aluminum coordination state and dynamics in those liquids. The present study re-enforces the current view that "minor" species such as penta-coordinated aluminum are actually present in a considerable amount in aluminosilicate glasses, and high-temperature liquids at and above the charge compensation join. The high-field strength of Y3+ and La3+ reveal, for the first time in glasses, a different mean electric field gradient perceived by the tetra- and penta-coordinated aluminum environments. The movements responsible for the NMR relaxation of aluminum in the high-temperature liquid are shown to be uncorrelated with the movements responsible for the macroscopic shear viscosity. Results obtained both on glasses and in situ at high-temperature suggest a preferential localization of Ln3+ nearby tetra-coordinated aluminum species, with possible formation of tricluster and/or Ln3+ coordination changes.

Formation of crosslinked PTFE by radiation-induced solid-state polymerization of tetrafluoroethylene at low temperatures

Tetrafluoroethylene monomer (TFE) was polymerized with gamma rays at various temperatures. The thermal properties of product were measured by using a differential scanning calorimeter (DSC) and the structure was analyzed by means of 19F high speed magic angle spinning nuclear magnetic resonance ( 19F NMR). It was found that, the PTFE obtained by the solid-state polymerization has been made clear to be crosslinked.

Solid‐state NMR characterization of 69Ga and 71Ga in crystalline solids

Gallium model systems containing four- and six-coordinate gallium sites have been investigated using solid-state NMR. Measurement of the isotropic chemical shift and electric field gradient (EFG) have been performed at 9.4 T on alpha-Ga2O3, beta-Ga2O3, LiGaO2, NaGaO2, KGaO2, Ga2(SO4)3, and LaGaO3 using a variety of techniques on both NMR active nuclei (69Ga and 71Ga) including static, high speed magic-angle spinning (MAS), satellite transition (ST) spectroscopy, and rotor-assisted population transfer (RAPT). The chemical shift is found to correlate well with the coordination number, with four-coordinate gallium having values of approximately 50 ppm and six-coordinate gallium having values near 225 ppm (referenced to 1 M gallium nitrate solution). The magnitude of the EFG is found to be correlated to the distortion of the gallium polyhedra, with the strained systems having EFGs of 3 x 10(21) Vm(-2) or more, while the less strained systems have values of 1.5 x 10(21) Vm(-2) or less. A plot of chemical shift versus EFG suggests that solid-state NMR of gallium oxyanions can be more discriminating than liquid state NMR chemical shifts alone.

Fluorine-19 solid state NMR study of vinylidenefluoride polymers using selective relaxation filters

Two fluoropolymers, poly(vinylidenefluoride) (PVDF) and a vinylidenefluoride telomer (VDFT), with molecular weights of 1×10 6 and 2×10 3 Da by GPC, respectively, have been analysed by 19F solid-state nuclear magnetic resonance (NMR) spectroscopy. Relaxation-filtered proton-decoupled magic-angle spinning (MAS) experiments, namely T 1 ρ filter, dipolar filter (DF), direct-polarisation delayed acquisition (DPDA) and discrimination induced by variable-amplitude minipulses (DIVAM), allowed signals in the direct polarisation (DP) spectra of PVDF and the VDFT to be discussed in terms of rigid and mobile domains. Both samples showed signals, which were multi-componential, but they differ in the nature of the crystalline form present. Thus, the Vinylidenefluoride (VDF) telomer exhibited a crystalline component corresponding to β PVDF, whereas the PVDF contained crystallites of the α form. Signals relating to end groups and reverse units, plus an anomalous signal displaying long-time transverse relaxation in the DPDA spectrum, were found for both polymers, though they showed diversity in chemical shift and content. Signals related to reverse units and/or end groups were seen between approximately −115 and approximately −117 ppm for both samples. High-speed MAS at higher magnetic field resulted in an increase in resolution so that signals previously attributed to single-phase characteristics are shown to indicate the possibility of several different mobilities. The results are debated with respect to molecular weight and relaxation parameters.

An effective stochastic excitation strategy for finding elusive NMR signals from solids

The versatility of using a stochastic pulse sequence to elucidate peaks with a wide range of shifts, peak widths, and T 1 relaxation times is demonstrated. A stochastic sequence is combined with high speed magic angle spinning (MAS) to obtain the broad and largely shifted peak associated with 31P in LiNiPO 4. A stochastic sequence is also used to obtain a spectrum of 85% H 3PO 4, which has a much longer T 1 value. The signal-to-noise was comparable for spectra of 85% H 3PO 4 obtained with either a stochastic sequence or an optimized Ernst angle experiment. Experimental parameters for the stochastic experiment are set depending only on the ringdown of the probe and not on any inherent qualities of the sample. A stochastic sequence, therefore, combined with MAS provides a useful strategy for finding peaks with unknown T 1 relaxation constants, peak widths, and shifts.

Sensitivity enhancement in two-dimensional solid-state NMR spectroscopy by transverse mixing.

The sensitivity of two-dimensional (2D) 13C-13C solid-state NMR spectroscopy under magic-angle spinning (MAS) is shown to be enhanced by the use of transverse polarization transfer in place of the conventional longitudinal polarization transfer. Experimental results are reported for 2D spectroscopy of a 20-residue, filament-forming peptide derived from the E. Coli RecA protein, containing five uniformly 13C-labeled residues, performed at 14.1 T with high-speed MAS and with finite-pulse radio-frequency-driven recoupling of dipolar interactions in the mixing period. Significant sensitivity enhancements observed at short mixing periods results from a more rapid build-up of cross-peaks under transverse mixing than under longitudinal mixing and from the 2 gain inherent in 2D measurements in which both orthogonal transverse polarization components in the t1 period contribute to each free-induction decay signal detected in the t2 period.

Sensitive high resolution inverse detection NMR spectroscopy of proteins in the solid state.

A new indirect detection scheme for obtaining (15)N/(1)H shift correlation spectra in crystalline proteins is described. Excellent water suppression is achieved without the need for pulsed field gradients, and using only a 2-step phase cycle. Careful attention to overall NMR instrument stability was found critical for obtaining the best resolution and sensitivity. Magnetic dilution by deuteration of the protein in combination with high-speed magic angle spinning produces (1)H resonances averaging only 0.22 ppm in width, and in some cases lines as narrow as 0.17 ppm are obtained. In application to two different polymorphs of ubiquitin, structure dependent differences in both (15)N and (1)H amide chemical shifts are observed. In one case, distinct shifts for different molecules in the asymmetric unit are seen, and all differ substantially from solution NMR shifts. A gain of 7 in sensitivity makes the method competitive with solution NMR as long as nanocrystalline samples are available.

Solid-state NMR spectroscopy method for determination of the backbone torsion angle psi in peptides with isolated uniformly labeled residues.

We demonstrate a solid-state nuclear magnetic resonance technique, with the acronym ROCSA-LG, for the determination of backbone torsion angles psi in peptides with multiple, but isolated, uniformly labeled residues. The method correlates the 13C' chemical shift anisotropy and the 13Calpha-1Halpha heteronuclear dipolar tensors within a single uniformly labeled residue in a two-dimensional (2D) experiment. The technique requires the measurement of only five 2D spectra and is compatible with high-speed magic-angle spinning. Experimental results are presented for the 17-residue alpha-helical peptide MB(i+4)EK and for amyloid fibrils formed by the 15-residue peptide Abeta11-25.

Constraints on supramolecular structure in amyloid fibrils from two-dimensional solid-state NMR spectroscopy with uniform isotopic labeling.

We show that strong constraints on supramolecular structure in amyloid fibrils can be obtained from solid-state nuclear magnetic resonance measurements on samples with uniformly 13C-labeled segments. The measurements exploit two-dimensional (2D) 13C-13C exchange spectroscopy in conjunction with high-speed magic angle spinning, with proton-mediated exchange of 13C nuclear spin magnetization as recently demonstrated by Baldus and co-workers (J. Am. Chem. Soc. 2002, 124, 9704-9705). Proton-mediated 2D exchange spectra of fibrils formed by residues 16-22 of the 40-residue Alzheimer's beta-amyloid peptide show strong nonsequential, intermolecular cross-peaks between alpha-carbons that dictate an antiparallel beta-sheet structure in which residue 16+k aligns with residue 22-k. The strong alpha/alpha cross-peaks are absent from conventional, direct 2D exchange spectra. Proton-mediated 2D exchange spectra of fibrils formed by residues 11-25 indicate an antiparallel beta-sheet structure with a pH-dependent intermolecular alignment. In contrast, proton-mediated 2D exchange spectra of fibrils formed by the full-length beta-amyloid peptide are consistent with a parallel beta-sheet structure. These data show that the supramolecular structure of amyloid fibrils is not determined by the amino acid sequence at the level of 7-residue or 15-residue segments. The proton-mediated 2D exchange spectra additionally demonstrate that the intermolecular alignment in the beta-sheets of these amyloid fibrils is highly ordered, with no detectable evidence for "misalignment" defects.

Recoupling of chemical shift anisotropies in solid-state NMR under high-speed magic-angle spinning and in uniformly C13-labeled systems

We demonstrate the possibility of recoupling chemical shift anisotropy (CSA) interactions in solid-state nuclear magnetic resonance (NMR) under high-speed magic-angle spinning (MAS) while retaining a static CSA powder pattern line shape and simultaneously attenuating homonuclear dipole–dipole interactions. CSA recoupling is accomplished by a rotation-synchronized radio-frequency pulse sequence with symmetry properties that permit static CSA line shapes to be obtained. We suggest a specific recoupling sequence, which we call ROCSA, for which the scaling factors for CSA and homonuclear dipole–dipole interactions are 0.272 and approximately 0.05, respectively. This sequence is suitable for high-speed C13 MAS NMR experiments on uniformly C13-labeled organic compounds, including biopolymers. We demonstrate the ROCSA sequence experimentally by measuring the C13 CSA patterns of the uniformly labeled, polycrystalline compounds L-alanine and N-acetyl-D,L-valine at MAS frequencies of 11 and 20 kHz. We also present experimental data for amyloid fibrils formed by a 15-residue fragment of the β-amyloid peptide associated with Alzheimer’s disease, in which four amino acid residues are uniformly labeled, demonstrating the applicability to biochemical systems of high molecular weight and significant complexity. Analysis of the CSA patterns in the amyloid fibril sample demonstrates the utility of ROCSA measurements as probes of peptide and protein conformation in noncrystalline solids.

Refinement of hydrogen atomic position in a hydrogen bond using a combination of solid-state NMR and computation.

DFT computations of the proton chemical shift for the intermolecular hydrogen bond in the white form of methylnitroacetanilide, together with the experimental value obtained by high-speed magic-angle spinning NMR, enable the N-H distance to be determined as 1.03 +/- 0.02 A.

Radiochemical Yields for Cross-Links and Branches in Radiation-Modified Poly(tetrafluoroethylene)

For the enhancement of the compatibility of fluoropolymers with other materials radiation modification is applied, for which irradiation with high-energy electrons provides an efficient and flexible possibility. Poly(tetrafluoroethylene) (PTFE) is known to predominantly undergo chain scission under irradiation. To counteract this chain scission and the resulting deterioration of the mechanical properties, PTFE has been irradiated at temperatures above the melting temperature under inert atmospheres. Under these conditions, in addition to chain scission long chain branches and cross-links are formed. The resulting structures have been studied by high-resolution fluorine-19 solid-state NMR applying highspeed magic-angle sample spinning. The structural changes as a function of energy dose and irradiation condition have been investigated upon irradiation under nitrogen atmosphere at 385 °C and under vacuum at 365 and 385 °C. Structures of interest are terminal groups, CF 3 side groups, branches, and cross-links. From quantitative information derived from the 19 F NMR spectra, the radiochemical yields, G(x) values, for the formation of CF 3 side groups, branches, and cross-links have been calculated. G(x) values of side groups and branches decrease with radiation dose, while the radiochemical yield of cross-links is nearly constant. These results are interpreted that in the first step in addition to main-chain scission the CF 3 side groups and branches were formed and in the second step after forming a sufficient quantity of side groups and branches the cross-links are formed.

Multiple Quantum 13C NMR Spectroscopy in Solids under High-Speed Magic-Angle Spinning

We demonstrate an approach to efficient excitation and detection of high-order multiple quantum (MQ) 13C NMR signals in solids under high-speed magic angle spinning (MAS). This approach combines ho...

High-Speed MAS 19F NMR Analysis of an Irradiated Fluoropolymer

The effect of gamma-radiation on a perfluoroalkoxy (PFA) resin was examined using solid-state high-speed magic angle spinning (MAS) F-19 NMR spectroscopy. Samples were prepared for analysis by subjecting them to gamma-radiation in the dose range 0.5-3 MGy at either 303, 473, or 573 K. New structures identified include new saturated chain ends, short and long branches, and unsaturated groups. The formation of branched structures was found to increase with increasing irradiation temperature; however, at all temperatures the radiation chemical yield (G value) of new chain ends was greater than the G value of long branch points, suggesting that chain scission is the net process.

Structural characterization of nylon 4 in the solid state by high-resolution solid-state 1H NMR spectroscopy

High-resolution solid-state 1H NMR spectra of nylon 4 melt-quenched sample and single crystal sample in the solid state were measured in a wide range of temperatures from room temperature to 505K by using solid state 300MHz NMR with the FSLG-2 homo-nuclear dipolar decoupling method combined with high speed magic angle spinning method. From the experimental results, structural characterization on the crystalline and non-crystalline components was carried out. Further, intermolecular interaction between nylon 4 and water contained in the sample was discussed.