Magnetic field gradients in solid state magic angle spinning NMR.

Magnetic field gradients have proven useful in NMR for coherence pathway selection, diffusion studies, and imaging. Recently they have been combined with magic angle spinning to permit high-resolution measurements of semi-solids, where magic angle spinning averages any residual dipolar couplings and local variations in the bulk magnetic susceptibility. Here we show the first examples of coherence pathway selection by gradients in dipolar coupled solids. When the gradient evolution competes with dipolar evolution the experiment design must take into account both the strength of the dipolar couplings and the means to refocus it. Examples of both homonuclear and heteronuclear experiments are shown in which gradients have been used to eliminate the need for phase cycling.     

A low-E magic angle spinning probe for biological solid state NMR at 750 MHz

Crossed-coil NMR probes are a useful tool for reducing sample heating for biological solid state NMR. In a crossed-coil probe, the higher frequency ¹H field, which is the primary source of sample heating in conventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltage is required, the electric field across the sample and the resultant heating is reduced. In this work we describe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual resonator. This dual resonator approach, referred to as “low-E,” was originally developed to reduce heating in samples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins in lipid bilayers, via MAS techniques requires large sample volumes at high field to obtain spectra with adequate signal-to-noise ratio under physiologically relevant conditions. With the low-E approach, we are able to obtain homogeneous and sufficiently strong radiofrequency fields for both ¹H and ¹³C frequencies in a 4 mm probe with a ¹H frequency of 750 MHz. The performance of the probe using windowless dipolar recoupling sequences is demonstrated on model compounds as well as membrane-embedded peptides.     

NMR imaging of solids with magic angle spinning.

Different aspects of solid state NMR imaging are reviewed, with emphasis on imaging in combination with line narrowing, especially in combination with magic angle spinning. Experimental results obtained with the latter technique are discussed, along with the implications of magic angle spinning on slice selection.     

15N spin diffusion rate in solid-state NMR of totally enriched proteins: the magic angle spinning frequency effect

As demonstrated by means of the one-dimensional solid-state MAS exchange experiment (CODEX), the rate of the proton driven spin diffusion between backbone (15)N nuclei in totally enriched protein depends strongly on the magic angle spinning (MAS) frequency: spin diffusion at MAS frequency 16 kHz is about 4-5 times slower as compared to that at MAS frequency 1 kHz which is due to the averaging of the homo- and hetero-nuclear dipolar interactions by MAS. It is important that even at the highest MAS frequencies used in our experiments the spin diffusion rate is comparable or larger than typical values of the spin-lattice relaxation rates of backbone nitrogens in solid proteins. Thus, the precise quantitative analysis of (15)N T(1)'s in totally enriched solid proteins may lead to wrong quantitative results. On the other hand, the effectiveness of the (15)N-(15)N correlation and structure determination experiments making use of (15)N-(15)N distances can be increased by decreasing the MAS frequency as far as possible, which is counter intuitive to the commonly applied fast MAS conditions in order to reduce the dipolar-broadened line widths for increased spectral resolution.     

Sensitivity enhancement, assignment, and distance measurement in 13C solid-state NMR spectroscopy for paramagnetic systems under fast magic angle spinning

Despite success of previous studies, high-resolution solid-state NMR (SSNMR) of paramagnetic systems has been still largely unexplored because of limited sensitivity/resolution and difficulty in assignment due to large paramagnetic shifts. Recently, we demonstrated that an approach using very-fast magic angle spinning (VFMAS; spinning speed 20kHz) enhances resolution/sensitivity in (13)C SSNMR for paramagnetic complexes [Y. Ishii, S. Chimon, N.P. Wickramasinghe, A new approach in 1D and 2D (13)C high resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning, J. Am. Chem. Soc. 125 (2003) 3438-3439]. In this study, we present a new strategy for sensitivity enhancement, signal assignment, and distance measurement in (13)C SSNMR under VFMAS for unlabeled paramagnetic complexes using recoupling-based polarization transfer. As a robust alternative of cross-polarization (CP), rapid application of recoupling-based polarization transfer under VFMAS is proposed. In the present approach, a dipolar-based analog of INEPT (dipolar INEPT) methods is used for polarization transfer and a (13)C signal is observed under VFMAS without (1)H decoupling. The resulting low duty factor permits rapid signal accumulation without probe arcing at recycle times ( approximately 3 ms/scan) matched to short (1)H T(1) values of small paramagnetic systems ( approximately 1 ms). Experiments on Cu(dl-Ala)(2) showed that the fast repetition approach under VFMAS provided sensitivity enhancement by a factor of 8-66 for a given sample, compared with the (13)C MAS spectrum under moderate MAS at 5kHz. The applicability of this approach was also demonstrated for a more challenging system, Mn(acac)(3), for which (13)C and (1)H paramagnetic shift dispersions reach 1500 and 700 ppm, respectively. It was shown that effective-evolution-time dependence of transferred signals in dipolar INEPT permitted one to distinguish (13)CH, (13)CH(2), (13)CH(3), (13)CO2- groups in 1D experiments for Cu(DL-Ala)(2) and Cu(Gly)(2). Applications of this technique to 2D (13)C/(1)H correlation NMR under VFMAS yielded reliable assignments of (1)H resonances as well as (13)C resonances for Cu(DL-Ala)(2) and Mn(acac)(3). Quantitative analysis of cross-peak intensities in 2D (13)C/(1)H correlation NMR spectra of Cu(DL-Ala)(2) provided distance information between non-bonded (13)C-(1)H pairs in the paramagnetic system.     

Sensitivity enhancement in structural measurements by solid state NMR through pulsed spin locking

Free induction decay (FID) signals in solid state NMR measurements performed with magic angle spinning can often be extended in time by factors on the order of 10 by a simple pulsed spin locking technique. The sensitivity of a structural measurement in which the structural information is contained in the dependence of the integrated FID amplitude on a preceding evolution period can therefore be enhanced substantially by pulsed spin locking in the signal detection period. We demonstrate sensitivity enhancements in a variety of solid state NMR techniques that are applicable to selectively isotopically labeled samples, including 13C-15N rotational echo double resonance (REDOR), 13C-13C dipolar recoupling measurements using the constant-time finite-pulse radio-frequency-driven recoupling (fpRFDR-CT) and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) techniques, and torsion angle measurements using the double quantum chemical shift anisotropy (DQCSA) technique. Further, we demonstrate that the structural information in the solid state NMR data is not distorted by pulsed spin locking in the detection period.     

15N chemical shift referencing in solid state NMR

Solid-state NMR spectroscopy has much advanced during the last decade and provides a multitude of data that can be used for high-resolution structure determination of biomolecules, polymers, inorganic compounds or macromolecules. In some cases the chemical shift referencing has become a limiting factor to the precision of the structure calculations and we have therefore evaluated a number of methods used in proton-decoupled ¹⁵N solid-state NMR spectroscopy. For ¹³C solid-state NMR spectroscopy adamantane is generally accepted as an external standard, but to calibrate the ¹⁵N chemical shift scale several standards are in use. As a consequence the published chemical shift values exhibit considerable differences (up to 22 ppm). In this paper we report the ¹⁵N chemical shift of several commonly used references compounds in order to allow for comparison and recalibration of published data and future work. We show that ¹⁵NH₄Cl in its powdered form (at 39.3 ppm with respect to liquid NH₃) is a suitable external reference as it produces narrow lines when compared to other reference compounds and at the same time allows for the set-up of cross-polarization NMR experiments. The compound is suitable to calibrate magic angle spinning and static NMR experiments. Finally the temperature variation of ¹⁵NH₄Cl chemical shift is reported.     

Field modulation effects induced by sample spinning: application to high-resolution magic angle spinning NMR

High-resolution magic angle spinning (HRMAS) has become an extremely versatile tool to study heterogeneous systems. HRMAS relies on magic angle spinning of the sample and on pulse sequences originally developed for liquid state NMR. In most cases the outcome of the experiment is conform to what is expected from high-resolution liquid state NMR spectroscopy. However in some instances, experiments run under MAS can produce some very puzzling results. After reviewing the basic hardware which is at the heart of HRMAS spectroscopy, we show that the origin of this behavior lies in the natural time-dependence of some physical quantities imparted by the rotation. We focus in particular on the effects of B1 inhomogeneities on the nutation, the (90 degrees)+x-t-(90 degrees )-x and the MLEV16 experiments. Different models of radiofrequency distribution of B1 fields in a solenoidal coil are derived from simple geometrical considerations. These models are shown by NMR spin dynamics calculations to reproduce the experimental NMR results. They are also consistent with electromagnetic simulations of the B1 field distribution inside a solenoidal coil.     

Structure determination of aligned systems by solid-state NMR magic angle spinning methods

Single crystal rotational echo double resonance (REDOR) experiments can be used to determine the three-dimensional orientation of heteronuclear bond vectors in an amino acid, as well as the crystal's orientation relative to the rotor fixed frame (RFF). We also demonstrate that for samples uniaxially aligned along the rotor axis, the polar tilt angle of a bond vector relative to the RFF can be measured by use of an analytical expression that describes the REDOR curve for that system. These bond orientations were verified by X-ray indexing of the single crystal sample, and were shown to be as accurate as +/- 1 degrees .     

Dynamics of the biopolymers in articular cartilage studied by magic angle spinning NMR

To understand the viscoelastic properties of cartilage tissue and for the development of tissue-engineered cartilage, we have studied the physicochemical properties of bovine nasal and pig articular cartilage by¹³C nuclear magnetic resonance (NMR) methods. The major macromolecular components of cartilage can be investigated individually by applying¹³C high-resolution (HR) NMR with scalar decoupling (for the polysaccharide component) and solid-state NMR with dipolar decoupling (for the collagen component). Partially resolved NMR spectra of the cartilage polysaccharides can be obtained by HR¹³C NMR indicating that these polysaccharides are highly mobile. Resonance lines have been assigned to chondroitin sulfate, the most mobile component of cartilage. To characterize time scales of molecular motions, we have measuredT ₁ andT ₂ relaxation times as a function of temperature and analyzed these data by means of a broad distribution of molecular correlation times. Typical correlation times for the large amplitude motions of chondroitin sulfate are of the order of 0.1–10 ns. For the detection and dynamical characterization of the cartilage collagen cross-polarization magic angle spinning (CP MAS) and high-power decoupling are indispensable.¹³C CP MAS spectra of cartilage are dominated by resonances from rigid collagen, while only low-intensity signals from the polysaccharides are observed. The good sensitivity at a magnetic field strength of 17.6 T allows the site-specific investigation of cartilage collagen dynamics by two-dimensional NMR methods. The cartilage collagen is essentially rigid with low-amplitude segmental motions on the fast time scale. Considering the high water content of cartilage and the almost isotropic mobility of the chondroitin sulfate molecules it is remarkable how little this affects the collagen dynamics. The dynamics of cartilage macromolecules is broadly distributed from almost completely rigid to highly mobile, which lends cartilage its mechanical strength and shock-absorbing properties.     

Pulsed field gradient magic angle spinning NMR self-diffusion measurements in liquids

Several investigations have recently reported the combined use of pulsed field gradient (PFG) with magic angle spinning (MAS) for the analysis of molecular mobility in heterogeneous materials. In contrast, little attention has been devoted so far to delimiting the role of the extra force field induced by sample rotation on the significance and reliability of self-diffusivity measurements. The main purpose of this work is to examine this phenomenon by focusing on pure liquids for which its impact is expected to be largest. Specifically, we show that self-diffusion coefficients can be accurately determined by PFG MAS NMR diffusion measurements in liquids, provided that specific experimental conditions are met. First, the methodology to estimate the gradient uniformity and to properly calibrate its absolute strength is briefly reviewed and applied on a MAS probe equipped with a gradient coil aligned along the rotor spinning axis, the so-called 'magic angle gradient' coil. Second, the influence of MAS on the outcome of PFG MAS diffusion measurements in liquids is investigated for two distinct typical rotors of different active volumes, 12 and 50 microL. While the latter rotor led to totally unreliable results, especially for low viscosity compounds, the former allowed for the determination of accurate self-diffusion coefficients both for fast and slowly diffusing species. Potential implications of this work are the possibility to measure accurate self-diffusion coefficients of sample-limited mixtures or to avoid radiation damping interferences in NMR diffusion measurements. Overall, the outlined methodology should be of interest to anyone who strives to improve the reliability of MAS diffusion studies, both in homogeneous and heterogeneous media.     

Sensitivity-enhanced static 15N NMR of solids by 1h indirect detection

A method for enhancing the sensitivity of 15N spectra of nonspinning solids through 1H indirect detection is introduced. By sampling the 1H signals in the windows of a pulsed spin-lock sequence, high-sensitivity 1H spectra can be obtained in two-dimensional (2D) spectra whose indirect dimension yields the 15N chemical shift pattern. By sacrificing the 1H chemical shift information, sensitivity gains of 1.8 to 2.5 for the 15N spectra were achieved experimentally. A similar sensitivity enhancement was also obtained for 2D (15)N-(1)H dipolar and 15N chemical shift correlation spectroscopy, by means of a 3D 1H/15N-1H/15N correlation experiment. We demonstrate this technique, termed PRINS for proton indirectly detected nitrogen static NMR, on a crystalline model compound with long 1H T(1rho) and on a 25-kDa protein with short 1H T(1rho). This 1H indirect detection approach should be useful for enhancing the sensitivity of 15N NMR of oriented membrane peptides. It can also be used to facilitate the empirical optimization of 15N-detected experiments where the inherent sensitivity of the sample is low.     

An approximate method of sideband intensity calculation in magic angle spinning NMR

A theory of sideband intensity is derived by expanding into a Taylor series the free induction decay observed under magic angle spinning (MAS). According to this procedure, the free induction decay signal is completely represented by a basis of irreducible tensors from rank zero to rank infinity. After averaging over all orientations, only the zero-order irreducible tensors contribute to the sideband intensities. Symmetry properties of the sidebands can be seen clearly in this expansion and an approximate formula up to ninth order is obtained by truncating the series. Sideband intensities can be calculated rapidly with this formula. The results agree satisfactorily with the exact sideband intensities obtained by numerical simulation if the ratio of the anisotropy to the spinning speed, ω₀δ/ω_r, is smaller than 3. The relationship of the sideband intensities with the moments of a MAS spectrum shows that the proposed method is an alternative to moment analysis when the spinning speed is not very slow. Anisotropic information about the chemical shift anisotropy interaction therefore can be extracted efficiently from experimental spectra by this approximate method.     

An accurate and simple method for setting the magic angle for solid state NMR studies

Use of the deuterium NMR spectrum of a deuteriated solute dissolved in the nematic phase of a liquid crystal is suggested for setting the magic angle in solid state NMR. The use of the method as a convenient and accurate means of setting the angle is demonstrated. Copyright 2000 Academic Press.     

Very fast magic angle spinning (1)H-(14)N 2D solid-state NMR: sub-micro-liter sample data collection in a few minutes

Substantial resolution and sensitivity enhancements of solid-state (1)H detected (14)N HMQC NMR spectra at very fast MAS rates up to 80 kHz, in a 1mm MAS rotor, are presented. Very fast MAS enhances the (1)H transverse relaxation time and efficiently decouples the (1)H-(14)N interactions, both effects leading to resolution enhancement. The micro-coil contributes to the sensitivity increase via strong (14)N rf fields and high sensitivity per unit volume. (1)H-(14)N HMQC 2D spectra of glycine and glycyl-L-alanine at 70 kHz MAS at 11.7 T are observed in a few minutes with a sample volume of 0.8 μL.     

In vivo detection of the cyclic osmoregulated periplasmic glucan of Ralstonia solanacearum by high-resolution magic angle spinning NMR.

We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.     

Microwave field distribution in a magic angle spinning dynamic nuclear polarization NMR probe

We present a calculation of the microwave field distribution in a magic angle spinning (MAS) probe utilized in dynamic nuclear polarization (DNP) experiments. The microwave magnetic field (B(1S)) profile was obtained from simulations performed with the High Frequency Structure Simulator (HFSS) software suite, using a model that includes the launching antenna, the outer Kel-F stator housing coated with Ag, the RF coil, and the 4mm diameter sapphire rotor containing the sample. The predicted average B(1S) field is 13μT/W(1/2), where S denotes the electron spin. For a routinely achievable input power of 5W the corresponding value is γ(S)B(1S)=0.84MHz. The calculations provide insights into the coupling of the microwave power to the sample, including reflections from the RF coil and diffraction of the power transmitted through the coil. The variation of enhancement with rotor wall thickness was also successfully simulated. A second, simplified calculation was performed using a single pass model based on Gaussian beam propagation and Fresnel diffraction. This model provided additional physical insight and was in good agreement with the full HFSS simulation. These calculations indicate approaches to increasing the coupling of the microwave power to the sample, including the use of a converging lens and fine adjustment of the spacing of the windings of the RF coil. The present results should prove useful in optimizing the coupling of microwave power to the sample in future DNP experiments. Finally, the results of the simulation were used to predict the cross effect DNP enhancement (?) vs. ω(1S)/(2π) for a sample of (13)C-urea dissolved in a 60:40 glycerol/water mixture containing the polarizing agent TOTAPOL; very good agreement was obtained between theory and experiment.     

Rotor synchronization of radiofrequency and gradient pulses in high-resolution magic angle spinning NMR

We have investigated the extent to which rotor synchronization of radiofrequency pulses leads to spectral improvement in high-resolution magic angle spinning NMR experiments. Several pulse sequences were tested, and the effect was found to be maximal in homonuclear TOCSY spectra. The physicochemical nature of the sample plays a role in the phenomenon, as rotor synchronization allows the refocusing of residual anisotropic interactions. However, even in a liquid sample the effects were visible. Radial inhomogeneities of the radiofrequency field were identified as an important source of the problem.     

Sensitivity enhancement in multiple-quantum NMR experiments with CPMG detection

We present a modified multiple-quantum (MQ) experiment, which implements the Carr-Purcell-Meiboom-Gill (CPMG) detection scheme in the static MQ NMR experiment proposed by W. S. Warren et al. (1980, J. Chem. Phys.73, 2084-2099) and exploited further by O. N. Antzutkin and R. Tycko (1999, J. Chem. Phys.110, 2749-2752). It is demonstrated that a significant enhancement in the sensitivity can be achieved by acquiring echo trains in the MQ experiments for static powder samples. The modified scheme employing the CPMG detection was superior to the original MQ experiment, in particular for the carbonyl carbon with a very large chemical shift anisotropy.