Sample Spinning Research Articles

Theoretical aspects of Magic Angle Spinning - Dynamic Nuclear Polarization

Magic Angle Spinning (MAS) combined with Dynamic Nuclear Polarization (DNP) has been proven in recent years to be a very powerful method for increasing solid-state NMR signals. Since the advent of biradicals such as TOTAPOL to increase the nuclear polarization new classes of radicals, with larger molecular weight and/or different spin properties have been developed. These have led to unprecedented signal gain, with varying results for different experimental parameters, in particular the microwave irradiation strength, the static field, and the spinning frequency. Recently it has been demonstrated that sample spinning imposes DNP enhancement processes that differ from the active DNP mechanism in static samples as upon sample spinning the DNP enhancements are the results of energy level anticrossings occurring periodically during each rotor cycle.In this work we present experimental results with regards to the MAS frequency dependence of the DNP enhancement profiles of four nitroxide-based radicals at two different sets of temperature, 110 and 160K. In fact, different magnitudes of reduction in enhancement are observed with increasing spinning frequency. Our simulation code for calculating MAS–DNP powder enhancements of small model spin systems has been improved to extend our studies of the influence of the interaction and relaxation parameters on powder enhancements. To achieve a better understanding we simulated the spin dynamics of a single three-spin system {ea-eb-n} during its steady state rotor periods and used the Landau–Zener formula to characterize the influence of the different anti-crossings on the polarizations of the system and their necessary action for reaching steady state conditions together with spin relaxation processes. Based on these model calculations we demonstrate that the maximum steady state nuclear polarization cannot become larger than the maximum polarization difference between the two electrons during the steady state rotor cycle. This study also shows the complexity of the MAS–DNP process and therefore the necessity to rely on numerical simulations for understanding parametric dependencies of the enhancements. Finally an extension of the spin system up to five spins allowed us to probe the first steps of the transfer of polarization from the nuclei coupled to the electrons to further away nuclei, demonstrating a decrease in the spin-diffusion barrier under MAS conditions.

The magnetic field dependence of cross-effect dynamic nuclear polarization under magic angle spinning.

We develop a theoretical description of Dynamic Nuclear Polarization (DNP) in solids under Magic Angle Spinning (MAS) to describe the magnetic field dependence of the DNP effect. The treatment is based on an efficient scheme for numerical solution of the Liouville-von Neumann equation, which explicitly takes into account the variation of magnetic interactions during the sample spinning. The dependence of the cross-effect MAS-DNP on various parameters, such as the hyperfine interaction, electron-electron dipolar interaction, microwave field strength, and electron spin relaxation rates, is analyzed. Electron spin relaxation rates are determined by electron paramagnetic resonance measurements, and calculations are compared to experimental data. Our results suggest that the observed nuclear magnetic resonance signal enhancements provided by MAS-DNP can be explained by discriminating between "bulk" and "core" nuclei and by taking into account the slow DNP build-up rate for the bulk nuclei.

Investigation of the Structure and Active Sites of TiO2 Nanorod Supported VOx Catalysts by High-Field and Fast-Spinning 51V MAS NMR

Supported VOx/TiO2-rod catalysts were studied by 51V MAS NMR at high field using a sample spinning rate of 55 kHz. The superior spectral resolution allows for the observation of at least five vanadate species. The assignment of these vanadate species was carried out by quantum chemical calculations of 51V NMR chemical shifts of model V surface structures. Methanol oxidative dehydrogenation (ODH) was used to establish a correlation between catalytic activity and the various surface V sites. It is found that monomeric V species are predominant at low vanadium loadings with two 51V NMR peaks observed at about −502 and −529 ppm. V dimers with two bridged oxygens result in a peak at about −555 ppm. Vanadate dimers and polyvanadates connected by one bridged oxygen atom between two adjacent V atoms resonate at about −630 ppm. A positive correlation is found between the V dimers, giving rise to the −555 ppm peak, and the ODH rate, and an even better correlation is obtained by including V monomer contributions. This result suggests that surface V dimers related to the −555 ppm peak and monomers are the primary active sites for the methanol ODH reaction. Furthermore, a portion of the V species is found to be invisible to NMR and the level of such invisibility increases with decreasing V loading levels, suggesting the existence of paramagnetic V species at the surface. These paramagnetic V species are also found to be much less active in methanol ODH.

A Decade of Experience With the UltraWide-Bore 900-MHz NMR Magnet

The ultrawide-bore (UWB) 900 MHz NMR magnet was commissioned in 2004. The magnet was conceived as part of the long range goal of NMR at fields in excess of 25 T that would require inner high temperature superconductor coils in the bore of a low temperature superconductor outer magnet of a sufficiently large bore. In order to address the combined interest in reaching 900 MHz while at the same time advancing the technology of large LTS NMR magnets, the decision was made to produce a 900 MHz magnet with an extended large bore to force the development of technology and for demonstration. Several advances in magnet technology were made in addition to the large bore in the areas of epoxy for impregnation and quench protection. During fabrication of the magnet, a joint between coil sections was possibly damaged, resulting in a large field drift in persistent mode. The cancelation of the field drift was initially a challenge but resulted in an excellent opportunity for the further development of drift compensation methods. Once the extended large bore was available, it was found to be increasingly of interest for unique NMR measurements. Since commissioning, the UWB magnet has played a central role in the NMR science program at the NHMFL. Major programs conducted in the magnet include biological solid state NMR both of uniformly oriented samples of membrane proteins that often represent drug targets for the pharmaceutical industry and magic angle sample spinning. The extended wide bore has allowed the magnet to be used for imaging at the high field of 21 T, including studies of high resolution 1 H images of adult rodents, 23 Na imaging in models ranging from single cells to in vivo rats to establish dynamic distributions of ionic sodium in pathological (stroke) and non-pathological (activation) conditions, and multi-modal nanoparticles used to track implanted cells as part of a cellular therapy for neurodegeneration. In addition 23 Na and 35 Cl images of in vivo tumors in animal models have suggested important potential roles for evaluating pharmaceutical efficacy and other medical applications.

New in situ solid-state NMR techniques for probing the evolution of crystallization processes: pre-nucleation, nucleation and growth.

The application of in situ techniques for investigating crystallization processes promises to yield significant new insights into fundamental aspects of crystallization science. With this motivation, we recently developed a new in situ solid-state NMR technique that exploits the ability of NMR to selectively detect the solid phase in heterogeneous solid-liquid systems (of the type that exist during crystallization from solution), with the liquid phase "invisible" to the measurement. As a consequence, the technique allows the first solid particles produced during crystallization to be observed and identified, and allows the evolution of different solid phases (e.g., polymorphs) present during the crystallization process to be monitored as a function of time. This in situ solid-state NMR strategy has been demonstrated to be a powerful approach for establishing the sequence of solid phases produced during crystallization and for the discovery of new polymorphs. The most recent advance of the in situ NMR methodology has been the development of a strategy (named "CLASSIC NMR") that allows both solid-state NMR and liquid-state NMR spectra to be measured (essentially simultaneously) during the crystallization process, yielding information on the complementary changes that occur in both the solid and liquid phases as a function of time. In this article, we present new results that highlight the application of our in situ NMR techniques to successfully unravel different aspects of crystallization processes, focusing on: (i) the application of a CLASSIC NMR approach to monitor competitive inclusion processes in solid urea inclusion compounds, (ii) exploiting liquid-state NMR to gain insights into co-crystal formation between benzoic acid and pentafluorobenzoic acid, and (iii) applications of in situ solid-state NMR for the discovery of new solid forms of trimethylphosphine oxide and L-phenylalanine. Finally, the article discusses a number of important fundamental issues relating to practical aspects, the interpretation of results and the future scope of these techniques, including: (i) an assessment of the smallest size of solid particle that can be detected in in situ solid-state NMR studies of crystallization, (ii) an appraisal of whether the rapid sample spinning required by the NMR measurement technique may actually influence or perturb the crystallization behaviour, and (iii) a discussion of factors that influence the sensitivity and time-resolution of in situ solid-state NMR experiments.

Pushing NMR sensitivity limits using dynamic nuclear polarization with closed-loop cryogenic helium sample spinning.

We report a strategy to push the limits of solid-state NMR sensitivity far beyond its current state-of-the-art. The approach relies on the use of dynamic nuclear polarization and demonstrates unprecedented DNP enhancement factors for experiments performed at sample temperatures much lower than 100 K, and can translate into 6 orders of magnitude of experimental time-savings. This leap-forward was made possible thanks to the employment of cryogenic helium as the gas to power magic angle sample spinning (MAS) for dynamic nuclear polarization (DNP) enhanced NMR experiments. These experimental conditions far exceed what is currently possible and allows currently reaching sample temperatures down to 30 K while conducting experiments with improved resolution (thanks to faster spinning frequencies, up to 25 kHz) and highly polarized nuclear spins. The impressive associated gains were used to hyperpolarize the surface of an industrial catalyst as well as to hyperpolarize organic nano-assemblies (self-assembling peptides in our case), for whom structures cannot be solved using diffraction techniques. Sustainable cryogenic helium sample spinning significantly enlarges the realm and possibilities of the MAS-DNP technique and is the route to transform NMR into a versatile but also sensitive atomic-level characterization tool.

(14)N overtone transition in double rotation solid-state NMR.

Solid-state NMR transitions involving outer energy levels of the spin-1 (14)N nucleus are immune, to first order in perturbation theory, to the broadening caused by the nuclear quadrupole interaction. The corresponding overtone spectra, when acquired in conjunction with magic-angle sample spinning, result in lines, which are just a few kHz wide, permitting the direct detection of nitrogen compounds without the need for labeling. Despite the success of this technique, "overtone" resonances are still broadened due to indirect, second order effects arising from the large quadrupolar interaction. Here we demonstrate that another order of magnitude in spectral resolution may be gained by using double rotation. This brings the width of the (14)N solid-state NMR lines much closer to the region commonly associated with high-resolution solid-state NMR spectroscopy of (15)N and demonstrates the improvements in resolution that may be possible through the development of pulsed methodologies to suppress these second order effects.

Proton decoupling and recoupling under double-nutation irradiation in solid-state NMR.

The effect of (1)H decoupling in magic-angle spinning solid-state NMR is studied under radiofrequency irradiation causing simultaneous nutations around a pair of orthogonal axes. Double-nutation with an arbitrary pair of nutation frequencies is implemented through modulation of the amplitude, phase, and frequency of the transmitting pulses. Similarity and difference of double-nutation decoupling and two-pulse phase-modulation decoupling schemes [A. E. Bennett, C. M. Rienstra, M. Auger, K. V. Lakshmi, and R. G. Griffin, J. Chem. Phys. 103, 6951-6958 (1995) and I. Scholz, P. Hodgkinson, B. H. Meier, and M. Ernst, J. Chem. Phys. 130, 114510 (2009)] are discussed. The structure of recoupling bands caused by interference of the (1)H spin nutation with sample spinning is studied by both experiments and numerical simulations.

Structural and interaction parameters of thermosensitive native α-elastin biohybrid microgel

The structural and water interaction parameters for native, α-elastin biohybrid microgel crosslinked with hydrophilic and hydrophobic crosslinkers are obtained from the volume phase transition temperature behaviour, 1H high-resolution magic-angle sample spinning transverse magnetization relaxation NMR, and modified Flory–Rehner swelling theory. Firstly, considering a homogeneous morphology the number of subchains in the biohybrid microgel, the residual water in deswollen state as a function of crosslink density and the temperature dependence of the Flory biopolymer–water interaction parameters are reported for the biohybrid microgels prepared with hydrophilic (PEG-DGE) and hydrophobic (BS3) crosslinkers. The Flory–Rehner classical approach is subsequently modified taking into account the heterogeneities observed by NMR transverse relaxation measurements. Two differently mobile regions are determined, a hydrophobic domain and a crosslinking domain with relative reduced mobility. For the first time, the influence of chain mobility on the Flory interaction parameter is investigated through a modified Flory state equation. The contributions of amino-acids located in the hydrophobic and crosslinking domains in the polypeptide sequence are separated while analyzing the biopolymer–water interaction.

Efficient heteronuclear decoupling in MAS solid-state NMR using non-rotor-synchronized rCW irradiation

We present new non-rotor-synchronized variants of the recently introduced refocused continuous wave (rCW) heteronuclear decoupling method significantly improving the performance relative to the original rotor-synchronized variants. Under non-rotor-synchronized conditions the rCW decoupling sequences provide more efficient decoupling, are easier to setup, and prove more robust towards experimental parameters such as radio frequency (rf) field amplitude and spinning frequency. This is demonstrated through numerical simulations substantiated with experimental results under different sample spinning and rf field amplitude conditions for powder samples of U-13C-glycine and U-13C-L-histidine·HCl·H2O.

Slow and Very Fast MAS Solid State NMR Study of Biopolymers

SummaryIn the first part of article, the “NMR Crystallography” approach as tool to fine refinement of solid state structure of biopolymers is presented, employing the α polymorph of L‐polylactide (PLLA) as model. Slow Magic Angle Spinning (MAS) technique (with spinning rate of sample in range from 1.2 kHz to 8.0 kHz) was used to assign 13C isotropic chemical shifts and values of 13C δii principal elements of chemical shifts tensors (CST). Theoretical 13C shielding parameters σii were obtained employing GIPAW (Gauge Invariant Projector Augmented Wave) method and compared with experimental 13C δii elements. The computed and experimental 13C CP/MAS spectra for WAND (Wide Angle Neutron Diffraction) geometry of powdered α PLLA were evaluated. It was revealed that the computed model of α PLLA model better fit to experimental NMR spectra.In the second part of article the applications of the new NMR methodology, so called very fast MAS (VF MAS) with sample spinning over 60 kHz are presented. The power of this approach is shown employing the 13C and 15N labeled protein, ubiquitin. We revealed that Cross‐Polarization with Variable Contact (CPVC) time sequence under very‐fast MAS condition performed in two‐dimensional (2D) mode is very efficient method to measure accurately the CH and NH distances, and to analyze the dynamics of proteins with overlapped resonances in aliphatic and aromatic regions.

Ibuprofen in Mesopores of Mobil Crystalline Material 41 (MCM-41): A Deeper Understanding

In this work, we compared two methods (incipient wetness and melting) for the encapsulation of ibuprofen in the pores of Mobil Crystalline Material 41 (MCM-41) through NMR (nuclear magnetic resonance) spectroscopy. (1)H NMR spectra were recorded under very fast MAS (sample spinning 60 kHz) conditions in both 1D and 2D mode (NOESY sequence). We also performed (13)C cross-polarization magic angle spinning (CP/MAS) experiments, (13)C single pulse experiments (SPE), and (1)H-(13)C HSQC HR/MAS (heteronuclear single quantum coherence high resolution) HR/MAS correlations. Evaluation of the encapsulation methods included an analysis of the filling factor of the drug into the pores. The stability of Ibu/MCM in an environment of ethanol or water vapor was tested. Our study showed that melting a mixture of Ibu and MCM is a much more efficient method of confining the drug in the pores compared to incipient wetness. The optimal experiments for the former method achieved a filling factor of approximately 60%. We concluded that the major limitation to the applicability of the incipient wetness method (filling factor ca. 20%) is the high affinity of solvent (typically ethanol) for MCM-41. We found that even ethanol vapor can remove Ibu from the pores. When a sample of Ibu/MCM was stored for a few hours in a closed vessel with ethanol vapor, Ibu was transported from the pores to the outer walls of MCM. We observed a similar phenomenon with water vapor, although this process is slower compared to the analogous procedure using ethanol. Our study clearly demonstrates that existing methods used to encapsulate drugs in mesoporous silica nanoparticles (MSNs) require reevaluation.

Detergent optimized membrane protein reconstitution in liposomes for solid state NMR.

For small helical membrane proteins, their structures are highly sensitive to their environment, and solid state NMR is a structural technique that can characterize these membrane proteins in native-like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation. A membrane protein from M. tuberculosis is used to illustrate the method. The results show that a detergent that stabilizes the most protein is not always ideal and sometimes cannot be removed by dialysis. By focusing on the lipid and protein binding properties of the detergent, proteoliposome preparations can be readily produced, which provide double the signal-to-noise ratios for both the oriented sample and magic angle spinning solid state NMR. The method will allow more membrane protein drug targets to be structurally characterized in lipid bilayer environments.

Pediatric dermatology: Part I

We present new non-rotor-synchronized variants of the recently introduced refocused continuous wave (rCW) heteronuclear decoupling method significantly improving the performance relative to the original rotor-synchronized variants. Under non-rotor-synchronized conditions the rCW decoupling sequences provide more efficient decoupling, are easier to setup, and prove more robust towards experimental parameters such as radio frequency (rf) field amplitude and spinning frequency. This is demonstrated through numerical simulations substantiated with experimental results under different sample spinning and rf field amplitude conditions for powder samples of U-13C-glycine and U-13C-L-histidine·HCl·H2O.

Structure of a Three Helix Membrane Protein from Oriented Sample and Magic Angle Spinning NMR Data

Of the many membrane protein structures deposited in the Protein Data Bank, only a few are of small helical proteins. Unfortunately, the typical transmembrane helix content of membrane proteins in the genomes of pathogenic bacteria is largely biased towards those with four or fewer helices. Many of these proteins are known or potential drug targets. The common structural techniques of x-ray crystallography and solution state NMR have had limited success in the determination of these structures [1]. Solid state NMR has emerged as a powerful tool capable of characterizing membrane protein structure in lipid bilayers. Oriented sample solid state NMR provides a set of peptide plane orientations. Magic angle spinning solid state NMR provides a set of interhelical distance restraints. Here, we show that together these restraints are capable of determining a backbone structure for the transmembrane domain of a three helix membrane protein. Furthermore, the structure orientation in the lipid bilayer is uniquely determined when using oriented sample restraints. We applied these methods to the Rv1861 protein from Mycobacterium tuberculosis, the causative agent of tuberculosis which is responsible for over one million deaths annually. Altogether, these experiments show that high resolution structures can be routinely determined for three to four helix membrane proteins and will help fill the void in the Protein Data Bank for these important drug targets.[1] Cross, T.A., Murray, D.T. and Watts, A. Eur. Biophys. J. In Press (2013) DOI 10.1007/s00249-013-0925-x.

Assessment of historic Tilia codrata wood by solid-state 13C CPMAS NMR spectroscopy

Abstract Historic lime ( Tilia cordata Mill.) wood samples, differing by their provenance, conservation status and period have been investigated by solid-state carbon-13 cross polarization magic angle sample spinning nuclear magnetic resonance ( 13 C CPMAS NMR) spectroscopy. Structural and chemical modifications were assessed by comparing the historic samples with a reference wood sample. The conventional NMR measurements followed by the 13 C resonance integral intensities of the wood samples have been carried out in order to acquire information of the chemical changes due to the natural ageing process taking place over the years. The main results concern the decrease of the carbohydrates moiety, especially the decrease of the hemicelluloses and amorphous cellulose signals, while the signals for aliphatic and methoxyl carbons from lignin present and increase of the intensity up to 120 years then start to decrease. At the same time a slight widening of the amorphous carbohydrate signals was observed, which may evidence the occurring of some chemical rearrangements, with the formation of new chemical species. These lead in the 13 C NMR spectra to the line broadening of the signals induced by their chemical shifts dispersion.

Experiments Optimized for Magic Angle Spinning and Oriented Sample Solid-State NMR of Proteins

Structure determination by solid-state NMR of proteins is rapidly advancing as a result of recent developments of samples, experimental methods, and calculations. There are a number of different solid-state NMR approaches that utilize stationary samples, aligned samples, or magic angle spinning of unoriented "powder" samples, and depending on the sample and the experimental method they can emphasize the measurement of distances or angles, ideally both, as sources of structural constraints. Multidimensional correlation spectroscopy of low-gamma nuclei such as (15)N and (13)C is an important step for making resonance assignments and measurements of angular restraints in membrane proteins. However, the efficiency of coherence transfer predominantly depends upon the strength of the dipole-dipole interaction, and this can vary from site to site and between sample alignments, for example, during the mixing of (13)C and (15)N magnetization in stationary aligned and in magic angle spinning samples. Here, we demonstrate that the efficiency of polarization transfer can be improved by using adiabatic demagnetization and remagnetization techniques on stationary aligned samples, and proton assisted insensitive nuclei cross-polarization in magic angle sample spinning samples. The adiabatic cross-polarization technique provides an alternative mechanism for spin-diffusion experiments correlating (15)N/(15)N and (15)N/(13)C chemical shifts over large distances. Improved efficiency in cross-polarization with 40-100% sensitivity enhancements is observed in proteins and single crystals, respectively. We describe solid-state NMR experimental techniques that are optimal for membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. The techniques are illustrated with data from single crystals both of peptides and of membrane proteins in phospholipid bilayers.

Grid-free powder averages: On the applications of the Fokker–Planck equation to solid state NMR

We demonstrate that Fokker–Planck equations in which spatial coordinates are treated on the same conceptual level as spin coordinates yield a convenient formalism for treating magic angle spinning NMR experiments. In particular, time dependence disappears from the background Hamiltonian (sample spinning is treated as an interaction), spherical quadrature grids are avoided completely (coordinate distributions are a part of the formalism) and relaxation theory with any linear diffusion operator is easily adopted from the Stochastic Liouville Equation theory. The proposed formalism contains Floquet theory as a special case. The elimination of the spherical averaging grid comes at the cost of increased matrix dimensions, but we show that this can be mitigated by the use of state space restriction and tensor train techniques. It is also demonstrated that low correlation order basis sets apparently give accurate answers in powder-averaged MAS simulations, meaning that polynomially scaling simulation algorithms do exist for a large class of solid state NMR experiments.

Full-length Vpu and human CD4(372–433) in phospholipid bilayers as seen by magic angle spinning NMR

HIV-1 Vpu and CD4(372-433), a peptide comprising the transmembrane and cytoplasmic domain of human CD4, were recombinantly expressed in Escherichia coli, uniformly labeled with 13C and 15N isotopes, and separately reconstituted into phospholipid bilayers. Highly resolved dipolar cross-polarization (CP)-based solid-state NMR spectra of the two transmembrane proteins were recorded under magic angle sample spinning. Partial assignment of 13C resonances was achieved. Site-specific assignments were obtained for 13 amino acid residues of CD4(372-433) and two Vpu residues. Additional amino acid type-specific assignments were achieved for 10 amino acid spin systems for both CD4(372-433) and Vpu. Further, structural flexibility was probed with different dipolar recoupling techniques, and the correct insertion of the transmembrane domains into the lipid bilayers was confirmed by proton spin diffusion experiments.

Membrane Protein Structure Determination in Membrana

The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure and the orientation of the protein within the membrane. When combined with dihedral angle restraints derived from analysis of isotropic chemical shifts, molecular fragment replacement, and de novo structure prediction, orientation restraints can yield high-quality three-dimensional structures with few or no distance restraints. Using complementary solid-state NMR methods based on oriented sample (OS) and magic angle spinning (MAS) approaches, one can resolve and assign multiple peaks through the use of (15)N/(13)C labeled samples and measure precise restraints to determine structures.