Slow Magic-angle Spinning Research Articles

Strategies for 1 H-Detected Dynamic Nuclear Polarization Magic-Angle Spinning NMR Spectroscopy.

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic‐angle spinning NMR spectroscopy. Herein, the feasibility of proton‐detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected 14N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.

Determination of Long-Range Distances by Fast Magic-Angle-Spinning Radiofrequency-Driven 19F-19F Dipolar Recoupling NMR.

Nanometer-range distances are important for restraining the three-dimensional structure and oligomeric assembly of proteins and other biological molecules. Solid-state NMR determination of protein structures typically utilizes 13C-13C and 13C-15N distance restraints, which can only be measured up to ∼7 Å because of the low gyromagnetic ratios of these nuclear spins. To extend the distance reach of NMR, one can harvest the power of 19F, whose large gyromagnetic ratio in principle allows distances up to 2 nm to be measured. However, 19F possesses large chemical shift anisotropies (CSAs) as well as large isotropic chemical shift dispersions, which pose challenges to dipolar coupling measurements. Here, we demonstrate 19F-19F distance measurements at high magnetic fields under fast magic-angle spinning (MAS) using radiofrequency-driven dipolar recoupling (RFDR). We show that 19F-19F cross-peaks for distances up to 1 nm can be readily observed in two-dimensional 19F-19F correlation spectra using less than 5 ms of RFDR mixing. This efficient 19F-19F dipolar recoupling is achieved using practically accessible MAS frequencies of 15-55 kHz, moderate 19F radio frequency field strengths, and no 1H decoupling. Experiments and simulations show that the fastest polarization transfer for aromatic fluorines with the highest distance accuracy is achieved using either fast MAS (e.g., 60 kHz) with large pulse duty cycles (>50%) or slow MAS with strong 19F pulses. Fast MAS considerably reduces relaxation losses during the RFDR π-pulse train, making finite-pulse RFDR under fast-MAS the method of choice. Under intermediate MAS frequencies (25-40 kHz) and intermediate pulse duty cycles (15-30%), the 19F CSA tensor orientation has a quantifiable effect on the polarization transfer rate; thus, the RFDR buildup curves encode both distance and orientation information. At fast MAS, the impact of CSA orientation is minimized, allowing pure distance restraints to be extracted. We further investigate how relayed transfer and dipolar truncation in multifluorine environments affect polarization transfer. This fast-MAS 19F RFDR approach is complementary to 19F spin diffusion for distance measurements and will be the method of choice under high-field fast-MAS conditions that are increasingly important for protein structure determination by solid-state NMR.

NMR longitudinal relaxation enhancement in metal halides by heteronuclear polarization exchange during magic-angle spinning.

Orders of magnitude decrease of (207)Pb and (199)Hg NMR longitudinal relaxation times T1 upon magic-angle-spinning (MAS) are observed and systematically investigated in solid lead and mercury halides MeX2 (Me = Pb, Hg and X = Cl, Br, I). In lead(ii) halides, the most dramatic decrease of T1 relative to that in a static sample is in PbI2, while it is smaller but still significant in PbBr2, and not detectable in PbCl2. The effect is magnetic-field dependent but independent of the spinning speed in the range 200-15 000 Hz. The observed relaxation enhancement is explained by laboratory-frame heteronuclear polarization exchange due to crossing between energy levels of spin-1/2 metal nuclei and adjacent quadrupolar-spin halogen nuclei. The enhancement effect is also present in lead-containing organometal halide perovskites. Our results demonstrate that in affected samples, it is the relaxation data recorded under non-spinning conditions that characterize the local properties at the metal sites. A practical advantage of fast relaxation at slow MAS is that spectral shapes with orientational chemical shift anisotropy information well retained can be acquired within a shorter experimental time.

Moderate MAS enhances local 1H spin exchange and spin diffusion

Proton NMR spin-diffusion experiments are often combined with magic-angle spinning (MAS) to achieve higher spectral resolution of solid samples. Here we show that local proton spin diffusion can indeed become faster at low (<10kHz) spinning rates as compared to static conditions. Spin diffusion under static conditions can thus be slower than the often referred value of 0.8nm2/ms, which was determined using slow MAS (Clauss et al., 1993). The enhancement of spin diffusion by slow MAS relies on the modulation of the orientation-dependent dipolar couplings during sample rotation and goes along with transient level crossings in combination with dipolar truncation. The experimental finding and its explanation is supported by density matrix simulations, and also emphasizes the sensitivity of spin diffusion to the local coupling topology. The amplification of spin diffusion by slow MAS cannot be explained by any model based on independent spin pairs; at least three spins have to be considered.

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.

Component-Specific Heterogeneity and Differential Phase Partitioning in Gradient Copolymers Revealed by Solids NMR

Gradient copolymers can exhibit physical properties that are different than their block polymer analogues. Property variations should depend upon differences in the molecular arrangement of individual comonomers in the polymer chain and in the gradient zone of each chain as well as the morphological arrangement of those chains in space. Here we describe experimental approaches based on fast and slow magic-angle spinning (MAS) NMR, which reveal the amount of rigid and soft phases in styrene–butadiene gradient copolymers with component specific resolution. In addition, we introduce a spin-counting strategy that quantitatively and directly determines the amount of the low-Tg, or “soft”, butadiene component that is incorporated into the rigid domains and also the amount of high-Tg, or “hard”, styrene component that is incorporated into the mobile domains. In total, the experiments provide bulk rigidity, amount of polybutadiene partitioned in both soft and hard phases and the amount of polystyrene partitioned in each phase. We show that the synthesis conditions can be changed to vary the partitioning of each gradient copolymer component in a systematic way and propose that the interphase between the hard and soft domains is responsible for differential partitioning.

Proton-detected 2D radio frequency driven recoupling solid-state NMR studies on micelle-associated cytochrome-b5

Solid-state NMR spectroscopy is increasingly used in the high-resolution structural studies of membrane-associated proteins and peptides. Most such studies necessitate isotopically labeled (13C, 15N and 2H) proteins/peptides, which is a limiting factor for some of the exciting membrane-bound proteins and aggregating peptides. In this study, we report the use of a proton-based slow magic angle spinning (MAS) solid-state NMR experiment that exploits the unaveraged 1H–1H dipolar couplings from a membrane-bound protein. We have shown that the difference in the buildup rates of cross-peak intensities against the mixing time – obtained from 2D 1H–1H radio frequency-driven recoupling (RFDR) and nuclear Overhauser effect spectroscopy (NOESY) experiments on a 16.7-kDa micelle-associated full-length rabbit cytochrome-b5 (cytb5) – can provide insights into protein dynamics and could be useful to measure 1H–1H dipolar couplings. The experimental buildup curves compare well with theoretical simulations and are used to extract relaxation parameters. Our results show that due to fast exchange of amide protons with water in the soluble heme-containing domain of cyb5, coherent 1H–1H dipolar interactions are averaged out for these protons while alpha and side chain protons show residual dipolar couplings that can be obtained from 1H–1H RFDR experiments. The appearance of resonances with distinct chemical shift values in 1H–1H RFDR spectra enabled the identification of residues (mostly from the transmembrane region) of cytb5 that interact with micelles.

Intramolecular Hydrogen Bonds in Cardiolipin

Cardiolipin was first isolated in 1941, and has since been implicated in several critical physiological roles. The distribution of cardiolipin species in tissues across prokaryotes and eukaryotes, and its concentration in membranes across which proton gradients drive energy production, is well characterized. The various functions attributed to cardiolipin include the structural stabilization of oxidative phosphorylation complexes in the inner mitochondrial membrane, where it constitutes about 25% of lipid. Its acid-base titration profile suggests a variable head group pKa in the physiological range, which leads to the hypothesis that cardiolipin also serves as a proton reservoir for energy-related protein complexes in the mitochondrial inter membrane space, thylakoid lumen, and at the outer leaflet of bacterial membranes. The importance of cardiolipin in these ascribed roles is further underscored by its involvement in diseases such as Barth's syndrome, where mitochondrial dysfunction is a major factor. In contrast to this physiological importance, Cardiolipin remains one of the least well characterised lipids from a biophysical perspective. In particular, there is still no consensus on whether it is a hydrogen bond between a protonated phosphate and the free 2-hydroxyl of the bridging glycerol of cardiolipin that explains its anomalous pKa. Furthermore, static 31P solid-state NMR characterisations of the head group have been difficult to interpret. We use our new Boltzmann-statistics maximum entropy analysis to globally analyse slow magic angle spinning spectra, and explain the uncharacteristic 31P static NMR spectra. Together with differential scanning calorimetry analysis, intriguing evidence emerges for the hydrogen bond hypothesis. We further pursue the hypothesis with additional solid-state NMR 31P{1H} heteronuclear correlations (HETCOR) and rotational-echo double-resonance (REDOR) experiments.

Chemical shift powder spectra enhanced by multiple-contact cross-polarization under slow magic-angle spinning

A simple multiple-contact cross-polarization (CP) scheme is applied to a powder sample of ferrocene and β-calcium formate under static and magic-angle spinning (MAS) conditions. The method is described analytically through the density matrix formalism. We show that multiple equilibrations–re-equilibrations with the proton spin bath improves the polarization transfer efficiency at short contact times and provides higher signal enhancements than state-of-the art techniques such as adiabatic passage through the Hartmann–Hahn condition CP (APHH-CP) when MAS is applied. The resulting chemical shift powder spectra then are identical to the ones obtained by using ROtor-Directed Exchange of Orientations CP (APHH-RODEO-CP) with intensity gains of a factor 1.1–1.3.

Chemical shift powder spectra obtained by using ROtor-Directed Exchange of Orientations Cross-Polarization (RODEO-CP)

We present a new simple and robust cross-polarization (CP) scheme, called ROtor-Directed Exchange of Orientations CP (RODEO-CP), which permits to obtain undistorted chemical shift powder spectra with short contact times, i.e., when a (quasi-)equilibrium state polarization is not reached. RODEO-CP uses slow magic-angle spinning to suppress lineshape distortions caused by the orientation dependence of CP. The reliability of the method is demonstrated on a powder sample of ferrocene and RODEO-CP is shown to remove the ‘magic-angle hole’ that distorts CP spectra of non-oriented membrane-associated peptides undergoing fast rotational diffusion around the bilayer normal.

Disentanglement of Heterogeneous Dynamics in Mixed Lipid Systems

Static phosphorous NMR has been a powerful technique for the study of model supramolecular phospholipid structures. Application to natural lipid bilayers with complex compositions, however, has been severely limited by the difficulty in deconvoluting overlapping broad lineshapes. We demonstrate a solution to this problem, using a global fit to a few slow magic-angle spinning spectra, in combination with an adaptation of Boltzmann statistics maximum entropy. The method provides a model-free means to characterize a heterogeneous mix of lipid dynamics via a distribution of 31P chemical shift anisotropies. It is used here to identify clear changes in membrane dynamics of a phosphatidylethanolamine and phosphatidylglycerol mixture, mimicking an Escherichia coli membrane upon addition of just 2% of the antimicrobial peptide maculatin 1.1. This illustration opens the prospect for investigation of arbitrarily complex natural lipid systems, important in many areas of biophysical chemistry and biomedicine.

Slow magic‐angle coil spinning: A high‐sensitivity and high‐resolution NMR strategy for microscopic biological specimens

High-resolution magic-angle spinning has become a valuable tool to study biologic samples; however, there are only a few high-resolution magic-angle spinning methodologies that have been explored specifically to deal with small semisolid biomaterials such as tissues. Here, we report a novel high-resolution (1)H NMR spectroscopic approach using magic-angle coil spinning. We demonstrate its potential for intact tissues by combining a resonant microcoil with a large-volume commercial magic-angle spinning rotor, where the microcoil offers incredible sensitivity and the large-volume magic-angle spinning rotor provides stable slow magic-angle spinning. This approach provides high-resolution spectra with high sensitivity and the preservation of living biologic specimens for an accurate chemical analysis.

A structure refinement strategy for NMR crystallography: An improved crystal structure of silica-ZSM-12 zeolite from 29Si chemical shift tensors

A strategy for performing crystal structure refinements with NMR chemical shift tensors is described in detail and implemented for the zeolite silica-ZSM-12 (framework type code MTW). The 29Si chemical shift tensors were determined from a slow magic-angle spinning spectrum obtained at an ultrahigh magnetic field of 21.1 T. The Si and O atomic coordinate parameters were optimized to give the best agreement between experimentally measured and ab initio calculated principal components of the 29Si chemical shift tensors, with the closest Si–O, O–O, and Si–Si distances restrained to correspond with the distributions of the distances found in a set of single-crystal X-ray diffraction (XRD) structures of high-silica zeolites. An improved structure for the silica-ZSM-12 zeolite, compared to a prior structure derived from powder XRD data, is obtained in which the agreement between the experimental and calculated 29Si chemical shift tensors is dramatically improved, the Si–O, O–O, and Si–Si distances correspond to the expected distributions, while the calculated powder XRD pattern remains in good agreement with the experimental powder XRD data. It is anticipated that this “NMR crystallography” structure refinement strategy will be an important tool for the accurate structure determination of materials that are difficult to fully characterize by traditional diffraction methods.

Probing Local Structure in Zeolite Frameworks: Ultrahigh-Field NMR Measurements and Accurate First-Principles Calculations of Zeolite 29Si Magnetic Shielding Tensors

The principal components of zeolite 29Si magnetic shielding tensors have been accurately measured and calculated for the first time. The experiments were performed at an ultrahigh magnetic field of 21.1 T in order to observe the small anisotropies of the 29Si shielding interactions that arise for Si atoms in near-tetrahedral geometries. A robust two-dimensional (2D) chemical shift anisotropy (CSA) recoupling pulse sequence was employed that enables quasi-static powder patterns to be resolved according to the isotropic chemical shifts. For the zeolites Sigma-2 and ZSM-12, it is demonstrated that the 29Si chemical shift (CS) tensor components measured by the recoupling experiment are in excellent agreement with those determined from spinning sidebands in slow magic-angle spinning (MAS) experiments. For the zeolite ZSM-5, the principal components of the 29Si CS tensors of 15 of the 24 Si sites were measured using the 2D CSA recoupling experiment, a feat that would not be possible with a slow MAS experiment due to the complexity of the spectrum. A simple empirical relationship between the 29Si CS tensors and local structural parameters could not be established. However, the 29Si magnetic shielding tensors calculated using Hartree-Fock ab initio calculations on clusters derived from the crystal structures are in excellent agreement with the experimental results. The accuracy of the calculations is strongly dependent on the quality of the crystal structure used in the calculation, indicating that the 29Si magnetic shielding interaction is extremely sensitive to the local structure around each Si atom. It is anticipated that the measurement and calculation of 29Si shielding tensors could be incorporated into the "NMR crystallography" of zeolites and other related silicate materials, possibly being used for structure refinements that may lead to crystal structures with very accurate Si and O atomic coordinates.

Multidimensional solid state NMR of anisotropic interactions in peptides and proteins

Accurate determinations of chemical shift anisotropy (CSA) tensors are valuable for NMR of biological systems. In this review we describe recent developments in CSA measurement techniques and applications, particularly in the context of peptides and proteins. These techniques include goniometeric measurements of single crystals, slow magic-angle spinning studies of powder samples, and CSA recoupling under moderate to fast MAS. Experimental CSA data can be analyzed by comparison with ab initio calculations for structure determination and refinement. This approach has particularly high potential for aliphatic (13)C analysis, especially Calpha tensors which are directly related to structure. Carbonyl and (15)N CSA tensors demonstrate a more complex dependence upon hydrogen bonding and electrostatics, in addition to conformational dependence. The improved understanding of these tensors and the ability to measure them quantitatively provide additional opportunities for structure determination, as well as insights into dynamics.

Solid-state phosphorus-31 NMR spectroscopy of a multiple-spin system: an investigation of a rhodium–triphosphine complex

Phosphorus-31 NMR spectra of solid [tris(dimethylphenylphosphine)](2,5-norbornadiene) rhodium(I) hexafluorophosphate have been acquired at several applied magnetic field strengths. The phosphorus nuclei of the three phosphine ligands are spin-spin coupled to each other and to 103Rh, resulting in complex NMR spectra; however, the three phosphorus chemical shift (CS) tensors were determined through the analysis of NMR spectra of slow magic angle spinning and stationary samples. Spectra of spinning samples in rotational resonance and two-dimensional 31P NMR spectra were particularly useful for determining the magnitudes of the indirect spin-spin couplings, and to probe their signs. Despite being in similar environments, the three phosphorus nuclei of the phosphine ligands have distinct CS tensors. In particular, the spans of these tensors, delta11-delta33, range from 80 to 176 ppm. The phosphorus CS tensors have been assigned to specific sites determined by X-ray crystallography, based on a combination of the experimental results and the results of quantum chemical calculations of the phosphorus shielding and 2J(31P,31P) values. The effect of coordination of dimethylphenylphosphine with rhodium has been investigated by comparing calculated phosphorus CS tensors for the uncoordinated ligand with those obtained for the ligands in the complex.

Determinations of 15N Chemical Shift Anisotropy Magnitudes in a Uniformly 15N,13C-Labeled Microcrystalline Protein by Three-Dimensional Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

Amide 15N chemical shift anisotropy (CSA) tensors provide quantitative insight into protein structure and dynamics. Experimental determinations of 15N CSA tensors in biologically relevant molecules have typically been performed by NMR relaxation studies in solution, goniometric analysis of single-crystal spectra, or slow magic-angle spinning (MAS) NMR experiments of microcrystalline samples. Here we present measurements of 15N CSA tensor magnitudes in a protein of known structure by three-dimensional MAS solid-state NMR. Isotropic 15N, 13C alpha, and 13C' chemical shifts in two dimensions resolve site-specific backbone amide recoupled CSA line shapes in the third dimension. Application of the experiments to the 56-residue beta1 immunoglobulin binding domain of protein G (GB1) enabled 91 independent determinations of 15N tensors at 51 of the 55 backbone amide sites, for which 15N-13C alpha and/or 15N-13C' cross-peaks were resolved in the two-dimensional experiment. For 37 15N signals, both intra- and interresidue correlations were resolved, enabling direct comparison of two experimental data sets to enhance measurement precision. Systematic variations between beta-sheet and alpha-helix residues are observed; the average value for the anisotropy parameter, delta (delta = delta(zz) - delta(iso)), for alpha-helical residues is 6 ppm greater than that for the beta-sheet residues. The results show a variation in delta of 15N amide backbone sites between -77 and -115 ppm, with an average value of -103.5 ppm. Some sites (e.g., G41) display smaller anisotropy due to backbone dynamics. In contrast, we observe an unusually large 15N tensor for K50, a residue that has an atypical, positive value for the backbone phi torsion angle. To our knowledge, this is the most complete experimental analysis of 15N CSA magnitude to date in a solid protein. The availability of previous high-resolution crystal and solution NMR structures, as well as detailed solid-state NMR studies, will enhance the value of these measurements as a benchmark for the development of ab initio calculations of amide 15N shielding tensor magnitudes.

Applications of the CSA-amplified PASS experiment

The recently reported CSA-amplified PASS experiment correlates the spinning sidebands at the true spinning frequency ω r with the spinning sidebands that would be obtained at the effective spinning frequency ω r / N , where N is termed the scaling factor. The experiment is useful for the measurement of small chemical shift anisotropies, for which slow magic-angle spinning frequencies, required to measure several spinning sidebands, can be unstable. We have experimentally evaluated the reliability of this experiment for this application. In particular we have demonstrated that large scaling factors of the order of N = 27 may be used, whilst still obtaining accurate chemical shift sideband intensities at the effective spinning frequency from the F 1 projection. Moreover, the sideband intensities are accurately obtained even in the presence of significant pulse imperfections. A second application of the CSA-amplified PASS experiment is the measurement of the chemical shift anisotropy of sites that experience homonuclear dipolar coupling, as may be found in uniformly labelled biological molecules, or for nuclei with a high natural abundance. The effects of homonuclear dipolar coupling on CSA-amplified PASS spectra has been investigated by numerical simulations and are demonstrated using uniformly 13C enriched l-histidine monohydrochloride monohydrate.

Origin of the High-Frequency Resonances in 1H NMR Spectra of Muscle Tissue: An in Vitro Slow Magic-Angle Spinning Study

High-resolution slow magic-angle spinning (150 Hz) 1H PASS NMR spectroscopy is performed on intact excised rat m. tibialis anterior. Untreated muscles and muscles in vitro incubated in Krebs-Ringers buffer based on deuterium oxide are investigated. In the high-frequency region of the 1H NMR spectra, resonances from H4 (approximately 7.1-7.2 ppm) and H2 (approximately 8.2-8.5 ppm) in histidine are observed. In addition, a resonance appears at 6.7 ppm for the untreated muscles. However, this resonance is absent in muscles following incubation in deuterium oxide. On the basis of its behavior in deuterium oxide combined with supplementary measurements for creatine solutions, the 6.7 ppm resonance is ascribed to the amino protons in creatine. Moreover, the present study demonstrates that the observation of the 6.7 ppm resonance depends on pH, which explains earlier reports stating its occasional appearance. Finally, measurements on solutions of ATP/AMP and histidine indicate that both ATP/AMP and histidine contribute to the resonances at approximately 8.2-8.5 ppm in the 1H NMR spectra of muscle tissue.

Orientation and Pore-Forming Mechanism of a Scorpion Pore-Forming Peptide Bound to Magnetically Oriented Lipid Bilayers

The orientation and pore-forming mechanisms of pandinin 2 (pin2), an antimicrobial peptide isolated from venom of the African scorpion Pandinus imperator, bound to magnetically oriented lipid bilayers were examined by 31P and 13C solid-state, and 15N liquid-state NMR spectroscopy. 31P NMR measurements at various temperatures, under neutral and acidic conditions, showed that membrane lysis occurred only under acidic conditions, and at temperatures below the liquid crystal-gel phase transition of the lipid bilayers, after incubation for two days in the magnet. Differential scanning calorimetry measurements showed that pin2 induced negative curvature strain in lipid bilayers. The 13C chemical shift values of synthetic pin2 labeled at Gly 3, Gly 8, Leu 12, Phe 17, or Ser 18 under static or slow magic-angle spinning conditions, indicate that pin2 penetrates the membrane with its average helical axis perpendicular to the membrane surface. Furthermore, amide H-D exchange experiments of 15N-Ala 4, Gly 8, and Ala 9 triply-labeled pin2 suggest that this peptide forms oligomers and confirms that the N-terminal region creates membrane pores.