Magic-angle Spinning Solid-state NMR Research Articles

Magnesium analogues of aluminosilicate inorganic polymers (geopolymers) from magnesium minerals

Attempts to synthesise magnesium-containing analogues of aluminosilicate geopolymers from the 1:1 and 2:1 layer magnesiosilicate minerals chrysotile and talc, as well as the magnesium mineral sepiolite are reported. The effect of pre-treating these starting minerals by grinding and/or dehydroxylation was also investigated by XRD, 29Si and natural-abundance 25Mg solid-state magic angle spinning (MAS) NMR spectroscopy. The products from sepiolite most closely resembled an aluminosilicate geopolymer, setting at 40 °C to an X-ray amorphous product containing a broad characteristic 29Si MAS NMR resonance at −90 ppm. The 25Mg MAS NMR spectrum of this product also showed evidence that some of the Mg was located in tetrahedral sites, as expected for a conventional geopolymer. A similar 25Mg MAS NMR result was obtained for chrysotile, but talc proved to be extremely resistant to geopolymer synthesis, requiring treatment at 120 °C for 3 days to set to a friable material retaining the XRD and NMR characteristics of the original talc or its crystalline dehydroxylation products. This lack of reactivity may be related to the 2:1 layer-lattice talc structure, or to the fact that a suitably reactive amorphous product is not formed upon dehydroxylation.

14N–1H Heteronuclear Multiple-Quantum Correlation Magic-Angle Spinning NMR Spectroscopy of Organic Solids

Abstract 14N–1H heteronuclear multiple-quantum correlation (HMQC) solid-state magic-angle spinning (MAS) NMR spectra recorded at a 1H Larmor frequency of 850 MHz are presented for the dipeptide β-AspAla. A modified version of the pulse sequence presented by Gan et al. (Chem. Phys. Lett. 435 (2007) 163) that utilises rotary resonance recoupling (R3) at the n = 2 condition (ν 1 = 2ν R ) is employed. Spectra recorded with a short recoupling period (under 200 μs) show two correlation peaks corresponding to the NH and NH3 moieties in the dipeptide. The quadrupolar product, P Q = C Q √ [1 + (η Q 2/3)], is determined experimentally as 3.1 MHz (NH) and 1.0 MHz (NH3) by a comparison of the 14N and 15N isotropic chemical shifts which differ due to the isotropic second-order quadrupolar shift for the spin I = 1 14N nucleus. It is shown that the peak sensitivities increase markedly upon increasing the MAS frequency from 30 to 45 to 60 kHz due to a combination of the reduced residual dipolar broadening of the 1H resonances and a lengthening of the coherence lifetimes under R 3 recoupling. Increasing the recoupling period leads to the observation of additional peaks corresponding to longer range intra- and intermolecular NH proximities. Reasonable agreement is evident upon comparing the experimental build-up of correlation peak intensity to that observed for eight-spin density-matrix simulations.

Organic amine grafting on mesoporous silica as a hybrid catalyst for heterogeneous three-component one-pot reaction

The organic–inorganic hybrid catalyst was prepared by immobilizing 3-methylaminopropyl moiety onto mesoporous silica, MCM-41, and applied for solid base catalyst of three-component one-pot reaction, i.e. Knoevenagel condensation of aldehyde with the active methylene compound to yield an electron deficient alkene which is subjective to be reacted with nitromethane by Michael addition to form tri-substituted primary nitro compound. The catalyst was characterized by powder-XRD, TG–DTA, FT-IR, nitrogen adsorption–desorption measurement and solid-state cross polarization magic angle spinning (CP/MAS) NMR, and exhibited high catalytic activity in three-component one-pot reaction; however, with poor reusability.

Synthesis and Mechanical Properties of Organic–Inorganic Hybrid Materials from Lignin and Polysiloxanes

The preparation of silica-containing organic-inorganic hybrid materials composed of kraft lignin, alkoxysilanes, and organic linkers was investigated. 3-Glycidyloxypropyltrimethoxysilane, 3-(triethoxysilyl)propylisocyanate (IPTES), and bis(trimethoxysilyl)hexane were selected as the most promising linkers. The best materials obtained showed improved mechanical and thermal properties compared with lignin itself. The reaction of the hydroxyl groups with IPTES and the sol-gel reaction between the organic linker molecules were studied by attenuated total reflectance FTIR and solid-state ²⁹Si magic-angle spinning NMR spectroscopy. The homogeneous composition was demonstrated by electron microscopy and energy-dispersive X-ray spectroscopy mapping. The mechanical properties were investigated by microindentation and dynamic mechanical thermal analysis.

Vegetable oil reactions within wood studied by direct 13C excitation with 1H decoupling and magic-angle sample spinning (MAS) NMR

Despite having been used for ages to protect wood against the influence of outdoor elements, the chemistry of vegetable oils within wood is poorly known. We propose a method based on solid-state magic-angle sample spinning NMR to in situ characterize oil oxidation as well as its immobilization. To eliminate signal coming from wood molecules but to keep signal from the oil, direct 13C excitation is performed with low-power 1H decoupling during signal acquisition. To suppress the effect of anisotropic spin-interactions and magnetic field inhomogeneity, the sample is spun at the magic-angle. Mono- and polyunsaturated fatty acid derivatives show a difference in their oxidation process: the monounsaturated methyl oleate reacts with wood components and becomes immobilized while the polyunsaturated methyl linoleate becomes oxidized and form oligomers but does not seem to bind to wood. Linola® oil behaves as would be expected on the basis of its composition by monounsaturated and polyunsaturated chains. This method can be generalized to all coating treatments to characterize chemical pathways and reactions. A better understanding of coating effects on wood is a crucial step to design more efficient protective mixtures.

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively 13C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged 1H–13C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain 2H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.

Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy

Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state nuclear magnetic resonance. However, the paucity of >5 Å distance restraints, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogs of the protein of interest containing covalently-attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu2+ mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone 15N PREs corresponding to ~10-20 Å distances were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å.

ChemInform Abstract: Applications of High‐Resolution 1H Solid‐State NMR

AbstractReview: 310 refs.

Bond covalency in perovskite oxynitrides ATaO2N (A = Ca, Sr, Ba) studied by 14N NMR spectroscopy

Local geometry and bond ionicity around the nitride ions in simple perovskite oxynitrides ATaO2N (A = Ca, Sr, Ba) have been investigated by solid-state magic-angle spinning (MAS) NMR spectroscopy. From all three compounds, fairly sharp 14N NMR peaks were observed, suggestive of the symmetric coordination environment of nitride ions. The 14N chemical shifts of ATaO2N, δ = 269–272 ppm relative to NH4Cl (δ = 0 ppm), are correlated to the bond ionicity, based on the N−Ta bond distances and Ta–N–Ta bond angles determined from the Rietveld refinement of neutron diffraction patterns. The 1H NMR measured for BaTaO2N presented a peak corresponding to H2O, implying that the polycrystalline surface of present oxynitride phases is covered by hydroxide terminals.

Dual Acquisition Magic‐Angle Spinning Solid‐State NMR‐Spectroscopy: Simultaneous Acquisition of Multidimensional Spectra of Biomacromolecules

Fast data collection: a general method for dual data acquisition of multidimensional magic-angle spinning solid-state NMR experiments is presented. The method uses a simultaneous Hartmann-Hahn cross-polarization from (1)H to (13)C and (15)N nuclei and exploits the long-living (15)N polarization for parallel acquisition of two multidimensional experiments.

Structure Determination of a Membrane Protein in Proteoliposomes

An NMR method for determining the three-dimensional structures of membrane proteins in proteoliposomes is demonstrated by determining the structure of MerFt, the 60-residue helix-loop-helix integral membrane core of the 81-residue mercury transporter MerF. The method merges elements of oriented sample (OS) solid-state NMR and magic angle spinning (MAS) solid-state NMR techniques to measure orientation restraints relative to a single external axis (the bilayer normal) from individual residues in a uniformly (13)C/(15)N labeled protein in unoriented liquid crystalline phospholipid bilayers. The method relies on the fast (>10(5) Hz) rotational diffusion of membrane proteins in bilayers to average the static chemical shift anisotropy and heteronuclear dipole-dipole coupling powder patterns to axially symmetric powder patterns with reduced frequency spans. The frequency associated with the parallel edge of such motionally averaged powder patterns is exactly the same as that measured from the single line resonance in the spectrum of a stationary sample that is macroscopically aligned parallel to the direction of the applied magnetic field. All data are collected on unoriented samples undergoing MAS. Averaging of the homonuclear (13)C/(13)C dipolar couplings, by MAS of the sample, enables the use of uniformly (13)C/(15)N labeled proteins, which provides enhanced sensitivity through direct (13)C detection as well as the use of multidimensional MAS solid-state NMR methods for resolving and assigning resonances. The unique feature of this method is the measurement of orientation restraints that enable the protein structure and orientation to be determined in unoriented proteoliposomes.

Structural Analysis of Crosslinking Junctions of Vulcanized Natural Rubber by Field Gradient-Fast Magic Angle Spinning Solid-State NMR Spectroscopy

Structural Analysis of Crosslinking Junctions of Vulcanized Natural Rubber by Field Gradient-Fast Magic Angle Spinning Solid-State NMR Spectroscopy

NMR Structure Determination of the Membrane Protein MerF in Bilayers

A general NMR method for determining the three-dimensional structures of membrane proteins in phospholipid bilayers is illustrated with the backbone structure of the mercury transporter MerF from the bacterial mercury detoxification system. MerF has two hydrophobic trans membrane helices and is responsible for the transport of mercuric ions and methylmercury across the membrane, from MerP in the periplasm to MerA in the cytoplasm. MerA is mercuric reductase, the enzyme that reduces the toxic Hg(II) to Hg(0). The structure determination method applied to membrane proteins combines Oriented Sample (OS) solid-state NMR and Magic Angle Spinning (MAS) solid-state NMR methods to measure angular constraints for structure calculations. The three-dimensional protein structure determined in its native bilayer environment enables the interactions of the mercury-binding cysteines with the metal ions and other proteins to be described.

Structure and Dynamics of Membrane-Associated Hepatitis C Virus Protein P7 by NMR Spectroscopy

P7 is a 63-residue viroporin encoded by human hepatitis C virus (HCV). It has ion channel activity and plays essential roles in viral proliferation. The NMR results show that the protein consists of two transmembrane spanning regions connected by a cytoplasmic loop. The solution NMR structural data that has been acquired includes: hydrogen/deuterium exchange, paramagnetic relaxation enhancement, residual dipolar couplings, and bicelle ‘q-titration.’ These data demonstrate that the protein has a range of dynamic properties, and interestingly two segments in each of the membrane spanning helices display local motions, possibly related to function. Oriented Sample (OS) solid-state NMR spectra of p7 in aligned phospholipid bilayers provided the tilt angles of the helical segments, 25° and 10° to the membrane normal. Recent magic angle spinning (MAS) solid-state NMR data of p7 in liposomes are being used to determine the three-dimensional structure. It has been shown that known channel-blocking compounds inhibit the ion channel activity. This and the fact that knockout models of this protein inhibit infectivity of the virus indicate it may be an ideal target for future drugs. There are six major genotypes and over 100 relevant subtypes. Our current studies aim to investigate the structure and dynamic differences among the genotypes. In addition to studying different genotypes, specific mutations to the HCV J4 genotype were made in order to modulate the dynamics of the protein and obtain additional secondary structural information. These studies have the potential to identify the reasons why different genotypes have varying activity and drug binding affinities.

Probing intermolecular interactions and nitrogen protonation in pharmaceuticals by novel 15N-edited and 2D 14N-1H solid-state NMR

We report the applications of two novel magic-angle spinning (MAS) solid-state NMR methods, 1J15N-1H spectral editing and 2D 14N-1H HMQC, to the characterisation of nitrogen functional groups in two pharmaceutical compounds, cimetidine and tenoxicam. The 1J15N-1H spectral editing method can readily differentiate the number of protons directly bonded to a nitrogen site and is not susceptible to motional effects. This enables confirmation of proton transfer, therefore proving or disproving amine salt formation, which is of high significance to the properties of a drug. The recently developed 2D 14N-1H HMQC method can demonstrate the presence of specific hydrogen bonding interactions and thus aid in identifying molecular association. First-principles calculations of NMR chemical shifts and quadrupolar parameters using the GIPAW method were combined with experimental data to assist with spectral assignment and the identification of the hydrogen bonding motifs.

Preparation and Catalytic Performances of a Molecularly Imprinted Ru‐Complex Catalyst with an NH2 Binding Site on a SiO2 Surface

A catalyst surface with an active metal site, a shape-selective reaction space, and an NH(2) binding site for o-fluorobenzophenone was designed and prepared by the molecular imprinting of a supported metal complex on a SiO(2) surface. A ligand of a SiO(2)-supported Ru complex that has a similar shape to the product of o-fluorobenzophenone hydrogenation was used as a template. An NH(2) binding site for o-fluorobenzophenone was spatially arranged on the wall of a molecularly imprinted cavity with a similar shape to the template. The structures of the SiO(2)-supported and molecularly imprinted Ru catalysts were characterized in a step-by-step manner by means of solid-state magic angle spinning (MAS) NMR, XPS, UV/Vis, N(2) adsorption, XRF, and Ru K-edge EXAFS. The molecularly imprinted Ru catalyst exhibited excellent shape selectivity for the transfer hydrogenation of benzophenone derivatives. It was found that the NH(2) binding site on the wall of the molecularly imprinted cavity enhanced the adsorption of o-fluorobenzophenone, of which the reduction product was imprinted, whereas there was no positive effect in the case of o-methylbenzophenone, which cannot interact with the NH(2) binding site through hydrogen bonding.

A spectrometer designed for 6.7 and 14.1 T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission

A Dynamic Nuclear Polarisation (DNP) enhanced solid-state Magic Angle Spinning (MAS) NMR spectrometer operating at 6.7T is described and demonstrated. The 187GHz TE13 fundamental mode of the FU CW VII gyrotron is used as the microwave source for this magnetic field strength and 284MHz 1H DNP-NMR. The spectrometer is designed for use with microwave frequencies up to 395GHz (the TE16 second-harmonic mode of the gyrotron) for DNP at 14.1T (600MHz 1H NMR).The pulsed microwave output from the gyrotron is converted to a quasi-optical Gaussian beam using a Vlasov antenna and transmitted to the NMR probe via an optical bench, with beam splitters for monitoring and adjusting the microwave power, a ferrite rotator to isolate the gyrotron from the reflected power and a Martin–Puplett interferometer for adjusting the polarisation. The Gaussian beam is reflected by curved mirrors inside the DNP-MAS-NMR probe to be incident at the sample along the MAS rotation axis. The beam is focussed to a ∼1mm waist at the top of the rotor and then gradually diverges to give much more efficient coupling throughout the sample than designs using direct waveguide irradiation. The probe can be used in triple channel HXY mode for 600MHz 1H and double channel HX mode for 284MHz 1H, with MAS sample temperatures ⩾85K. Initial data at 6.7T and ∼1W pulsed microwave power are presented with 13C enhancements of 60 for a frozen urea solution (1H–13C CP), 16 for bacteriorhodopsin in purple membrane (1H–13C CP) and 22 for 15N in a frozen glycine solution (1H–15N CP) being obtained. In comparison with designs which irradiate perpendicular to the rotation axis the approach used here provides a highly efficient use of the incident microwave beam and an NMR-optimised coil design.

Similarities and Differences within Members of the Ff Family of Filamentous Bacteriophage Viruses

The filamentous bacteriophage viruses of the Ff family, fd and M13, slightly differ in their genome, and their 50-residue-long major capsid proteins have a single site difference: the uncharged asparagine-12 in M13 is replaced with a negatively charged aspartate in fd. We have used magic-angle spinning solid-state NMR spectroscopy to site-specifically assign the resonances belonging to the capsid protein of M13. Assignment of several mobile residues was facilitated by using J-based spectroscopy, which in addition provided sugar-base contacts in the M13-DNA stemming from two-bond scalar couplings. A comparison between M13 and fd bacteriophages reveals that the two virions have a very conserved and stable structure, manifested in negligibly small chemical shift differences and similar dynamic properties for nearly all resonances. The principal difference between the two phages involves residues in the vicinity of residue 12. We suggest that the elimination of the single charge at position 12 throughout the entire assembly affects the electrostatic and hydrogen-bonding interaction network governing inter- and intraresidue contacts, mainly by the rearrangement of the positively charged lysine residue at position 8.

Solid-state magic-angle spinning NMR of membrane proteins and protein–ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future ‘big’ projects.

A software framework for analysing solid-state MAS NMR data.

Solid-state magic-angle-spinning (MAS) NMR of proteins has undergone many rapid methodological developments in recent years, enabling detailed studies of protein structure, function and dynamics. Software development, however, has not kept pace with these advances and data analysis is mostly performed using tools developed for solution NMR which do not directly address solid-state specific issues. Here we present additions to the CcpNmr Analysis software package which enable easier identification of spinning side bands, straightforward analysis of double quantum spectra, automatic consideration of non-uniform labelling schemes, as well as extension of other existing features to the needs of solid-state MAS data. To underpin this, we have updated and extended the CCPN data model and experiment descriptions to include transfer types and nomenclature appropriate for solid-state NMR experiments, as well as a set of experiment prototypes covering the experiments commonly employed by solid-sate MAS protein NMR spectroscopists. This work not only improves solid-state MAS NMR data analysis but provides a platform for anyone who uses the CCPN data model for programming, data transfer, or data archival involving solid-state MAS NMR data.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9569-2) contains supplementary material, which is available to authorized users.