Ultrafast Magic Angle Spinning Research Articles

Nano-mole scale side-chain signal assignment by 1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling.

We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.

Recovery of bulk proton magnetization and sensitivity enhancement in ultrafast magic-angle spinning solid-state NMR.

The sensitivity of solid-state NMR experiments is limited by the proton magnetization recovery delay and by the duty cycle of the instrument. Ultrafast magic-angle spinning (MAS) can improve the duty cycle by employing experiments with low-power radio frequency (RF) irradiation which reduce RF heating. On the other hand, schemes to reduce the magnetization recovery delay have been proposed for low MAS rates, but the enhancements rely on selective transfers where the bulk of the (1)H magnetization pool does not contribute to the transfer. We demonstrate here that significant sensitivity enhancements for selective and broadband experiments are obtained at ultrafast MAS by preservation and recovery of bulk (1)H magnetization. We used [(13)C, (15)N]-labeled glutamine as a model compound, spinning in a 1.3 mm rotor at a MAS frequency of 65 kHz. Using low-power (1)H RF (13.4 kHz), we obtain efficient (1)H spin locking and (1)H-(13)C decoupling at ultrafast MAS. As a result, large amounts of (1)H magnetization, from 35% to 42% of the initial polarization, are preserved after cross-polarization and decoupling. Restoring this magnetization to the longitudinal axis using a flip-back pulse leads to an enhancement of the sensitivity, an increase ranging from 14% to 21% in the maximal achievable sensitivity regime and from 24% to 50% in the fast pulsing regime, and to a shortening of the optimal recycling delay to 68% of its original duration. The analysis of the recovery and sensitivity curves reveals that the sensitivity gains do not rely on a selective transfer where few protons contribute but rather on careful conservation of bulk (1)H magnetization. This makes our method compatible with broadband experiments and uniformly labeled materials, in contrast to the enhancement schemes proposed for low MAS. We tested seven different cross-polarization schemes and determined that recovery of bulk (1)H magnetization is a general method for sensitivity enhancement. The physical insight gained about the behavior of proton magnetization sharing under spin lock will be helpful to break further sensitivity boundaries, when even higher external magnetic fields and faster spinning rates are employed.

Investigating albendazole desmotropes by solid-state NMR spectroscopy.

Characterization of the molecular structure and physicochemical solid-state properties of the solid forms of pharmaceutical compounds is a key requirement for successful commercialization as potential active ingredients in drug products. These properties can ultimately have a critical effect on the solubility and bioavailability of the final drug product. Here, the desmotropy of Albendazole forms I and II was investigated at the atomic level. Ultrafast magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy, together with powder X-ray diffraction, thermal analysis, and Fourier transform infrared spectroscopy, were performed on polycrystalline samples of the two solids in order to fully characterize and distinguish the two forms. High-resolution one-dimensional (1)H, (13)C, and (15)N together with two-dimensional (1)H/(1)H single quantum-single quantum, (1)H/(1)H single quantum-double quantum, and (1)H/(13)C chemical shift correlation solid-state NMR experiments under MAS conditions were extensively used to decipher the intramolecular and intermolecular hydrogen bonding interactions present in both solid forms. These experiments enabled the unequivocal identification of the tautomers of each desmotrope. Our results also revealed that both solid forms may be described as dimeric structures, with different intermolecular hydrogen bonds connecting the tautomers in each dimer.

Observation of 1H–13C and 1H–1H proximities in a paramagnetic solid by NMR at high magnetic field under ultra-fast MAS

The assignment of NMR signals in paramagnetic solids is often challenging since: (i) the large paramagnetic shifts often mask the diamagnetic shifts specific to the local chemical environment, and (ii) the hyperfine interactions with unpaired electrons broaden the NMR spectra and decrease the coherence lifetime, thus reducing the efficiency of usual homo- and hetero-nuclear NMR correlation experiments. Here we show that the assignment of 1H and 13C signals in isotopically unmodified paramagnetic compounds with moderate hyperfine interactions can be facilitated by the use of two two-dimensional (2D) experiments: (i) 1H–13C correlations with 1H detection and (ii) 1H–1H double-quantum↔single-quantum correlations. These methods are experimentally demonstrated on isotopically unmodified copper (II) complex of l-alanine at high magnetic field (18.8T) and ultra-fast Magic Angle Spinning (MAS) frequency of 62.5kHz. Compared to 13C detection, we show that 1H detection leads to a 3-fold enhancement in sensitivity for 1H–13C 2D correlation experiments. By combining 1H–13C and 1H–1H 2D correlation experiments with the analysis of 13C longitudinal relaxation times, we have been able to assign the 1H and 13C signals of each l-alanine ligand.

Composite-180° pulse-based symmetry sequences to recouple proton chemical shift anisotropy tensors under ultrafast MAS solid-state NMR spectroscopy

There is considerable interest in the measurement of proton (1H) chemical shift anisotropy (CSA) tensors to obtain deeper insights into H-bonding interactions which find numerous applications in chemical and biological systems. However, the presence of strong 1H/1H dipolar interaction makes it difficult to determine small size 1H CSAs from the homogeneously broadened NMR spectra. Previously reported pulse sequences for 1H CSA recoupling are prone to the effects of radio frequency field (B1) inhomogeneity. In the present work we have carried out a systematic study using both numerical and experimental approaches to evaluate γ-encoded radio frequency (RF) pulse sequences based on R-symmetries that recouple 1H CSA in the indirect dimension of a 2D 1H/1H anisotropic/isotropic chemical shift correlation experiment under ultrafast magic angle spinning (MAS) frequencies. The spectral resolution and sensitivity can be significantly improved in both frequency dimensions of the 2D 1H/1H correlation spectrum without decoupling 1H/1H dipolar couplings but by using ultrafast MAS rates up to 70kHz. We successfully demonstrate that with a reasonable RF field requirement (<200kHz) a set of symmetry-based recoupling sequences, with a series of phase-alternating 270°0–90°180 composite-180° pulses, are more robust in combating B1 inhomogeneity effects. In addition, our results show that the new pulse sequences render remarkable 1H CSA recoupling efficiency and undistorted CSA lineshapes. Experimental results on citric acid and malonic acid comparing the efficiencies of these newly developed pulse sequences with that of previously reported CSA recoupling pulse sequences are also reported under ultrafast MAS conditions.

Insights into the Structure and Dynamics of Measles Virus Nucleocapsids by 1H-detected Solid-state NMR

1H-detected solid-state nuclear magnetic resonance (NMR) experiments are recorded on both intact and trypsin-cleaved sedimented measles virus (MeV) nucleocapsids under ultra-fast magic-angle spinning. High-resolution 1H,15N-fingerprints allow probing the degree of molecular order and flexibility of individual capsid proteins, providing an exciting atomic-scale complement to electro microscopy (EM) studies of the same systems.

Solid-state NMR structure characterization of a 13CO-Labeled Ir(I) complex with a P,N-donor ligand including ultrafast MAS methods.

The structural characterization of a (13)CO-labeled Ir(I) complex bearing an P,N-donor ligand (1-[2-(diphenylphosphino)ethyl]pyrazole), [Ir(PyP)((13)CO)Cl] is demonstrated using a series of tailored solid-state NMR techniques based on ultrafast (60 kHz) Magic Angle Spinning (MAS), which facilitates correlations with narrow proton line-widths. Our 1D (1)H MAS and 2D (13)C and (31)P CP-MAS NMR spectra provided structural information similar to that obtained using NMR spectroscopy in solution. We employed high-resolution 2D solid-state correlation spectroscopy ((1)H-(13)C HETCOR, (1)H-(31)P correlation) to characterize the networks of dipolar couplings between protons and carbon/phosphorus. (1)H-(1)H SQ-SQ correlation spectra showed the dipolar contacts between all protons in a similar fashion to its solution counterpart, NOESY. The use of the (1)H single quantum/double quantum experiments made it possible to observe the dipolar-coupling contacts between immediately adjacent protons. Additionally, internuclear (13)CO-(31)P distance measurements were performed using REDOR. The combination of all of these techniques made it possible to obtain comprehensive structural information on the molecule [Ir(PyP)((13)CO)Cl] in the solid state, which is in excellent agreement with the single crystal X-ray structure of the complex, and demonstrates the enormous value of ultrafast MAS NMR techniques for a broad range of future applications.

ChemInform Abstract: Ultrafast Magic Angle Spinning Nuclear Magnetic Resonance

ChemInformVolume 45, Issue 28 Reviews ChemInform Abstract: Ultrafast Magic Angle Spinning Nuclear Magnetic Resonance Michael Deschamps, Michael Deschamps Cond. Extremes Mater. Haute Temp. Irradiat., CNRS, Univ. Orleans, F-45071 Orleans, Fr.Search for more papers by this author Michael Deschamps, Michael Deschamps Cond. Extremes Mater. Haute Temp. Irradiat., CNRS, Univ. Orleans, F-45071 Orleans, Fr.Search for more papers by this author First published: 26 June 2014 https://doi.org/10.1002/chin.201428297Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume45, Issue28July 15, 2014 RelatedInformation

Investigating Local Structure in Layered Double Hydroxides with 17O NMR Spectroscopy

A new method based on the “memory effect” for efficient and economical 17O labeling of layered double hydroxides (LDHs) is introduced. High‐quality 17O solid‐state NMR spectra are obtained, for the first time, for LDHs prepared with only several hundred microliters of 17O‐enriched H2O. The 17O resonances due to the different oxygen ions in the structure of LDHs can be resolved with better resolution than the results obtained from 1H ultrafast magic angle spinning (MAS) NMR spectroscopy. The results show clear evidence for Al–O–Al avoidance. Since only intermediate MAS speeds and fields are used, this new approach can be incorporated easily with a variety of dipolar recoupling schemes to explore the key interactions and applications of LDHs.

Sensitivity and Resolution Enhanced Solid-State NMR for Paramagnetic Systems and Biomolecules under Very Fast Magic Angle Spinning

Recent research in fast magic angle spinning (MAS) methods has drastically improved the resolution and sensitivity of NMR spectroscopy of biomolecules and materials in solids. In this Account, we summarize recent and ongoing developments in this area by presenting (13)C and (1)H solid-state NMR (SSNMR) studies on paramagnetic systems and biomolecules under fast MAS from our laboratories. First, we describe how very fast MAS (VFMAS) at the spinning speed of at least 20 kHz allows us to overcome major difficulties in (1)H and (13)C high-resolution SSNMR of paramagnetic systems. As a result, we can enhance both sensitivity and resolution by up to a few orders of magnitude. Using fast recycling (∼ms/scan) with short (1)H T1 values, we can perform (1)H SSNMR microanalysis of paramagnetic systems on the microgram scale with greatly improved sensitivity over that observed for diamagnetic systems. Second, we discuss how VFMAS at a spinning speed greater than ∼40 kHz can enhance the sensitivity and resolution of (13)C biomolecular SSNMR measurements. Low-power (1)H decoupling schemes under VFMAS offer excellent spectral resolution for (13)C SSNMR by nominal (1)H RF irradiation at ∼10 kHz. By combining the VFMAS approach with enhanced (1)H T1 relaxation by paramagnetic doping, we can achieve extremely fast recycling in modern biomolecular SSNMR experiments. Experiments with (13)C-labeled ubiquitin doped with 10 mM Cu-EDTA demonstrate how effectively this new approach, called paramagnetic assisted condensed data collection (PACC), enhances the sensitivity. Lastly, we examine (13)C SSNMR measurements for biomolecules under faster MAS at a higher field. Our preliminary (13)C SSNMR data of Aβ amyloid fibrils and GB1 microcrystals acquired at (1)H NMR frequencies of 750-800 MHz suggest that the combined use of the PACC approach and ultrahigh fields could allow for routine multidimensional SSNMR analyses of proteins at the 50-200 nmol level. Also, we briefly discuss the prospects for studying bimolecules using (13)C SSNMR under ultrafast MAS at the spinning speed of ∼100 kHz.

Out-and-back 13C–13C scalar transfers in protein resonance assignment by proton-detected solid-state NMR under ultra-fast MAS

We present here (1)H-detected triple-resonance H/N/C experiments that incorporate CO-CA and CA-CB out-and-back scalar-transfer blocks optimized for robust resonance assignment in biosolids under ultra-fast magic-angle spinning (MAS). The first experiment, (H)(CO)CA(CO)NH, yields (1)H-detected inter-residue correlations, in which we record the chemical shifts of the CA spins in the first indirect dimension while during the scalar-transfer delays the coherences are present only on the longer-lived CO spins. The second experiment, (H)(CA)CB(CA)NH, correlates the side-chain CB chemical shifts with the NH of the same residue. These high sensitivity experiments are demonstrated on both fully-protonated and 100%-H(N) back-protonated perdeuterated microcrystalline samples of Acinetobacter phage 205 (AP205) capsids at 60 kHz MAS.

13C‐Detected Through‐Bond Correlation Experiments for Protein Resonance Assignment by Ultra‐Fast MAS Solid‐State NMR

We present two sequences which combine ((1)H,(15)N) and ((15)N,(13)C) selective cross-polarization steps with an efficient variant of the J-based homonuclear transfer scheme, in which a spin-state-selective (S(3)E) block is incorporated to improve both resolution and sensitivity in the direct (13)C dimension. We propose these two sequences as a part of a suite of four N-C correlation experiments allowing for the assignment of protein backbone resonances in the solid state. We illustrate these experiments under ultra-fast magic angle spinning conditions on two samples of microcrystalline dimeric human superoxide dismutase (SOD, 153×2 amino acids), in its diamagnetic ("empty", Zn(II)) and paramagnetic (Cu(II), Zn(II)) states.

Frequency-stepped acquisition in nuclear magnetic resonance spectroscopy under magic angle spinning

The nuclear magnetic resonance of paramagnetic solids is usually characterized by the presence of large chemical shifts and shift anisotropies due to hyperfine interactions. Frequently the resulting spectra cover a frequency range of several megahertz, which is greater than the bandwidth of commercially available radio-frequency (RF) probes, making it impossible to acquire the whole spectrum in a single experiment. In these cases it common to record a series of spectra, in which the probe is tuned to a different frequency for each, and then sum the results to give the "true" spectrum. While this method is very widely used on static samples, the application of frequency stepping under magic-angle spinning (MAS) is less common, owing to the increased complexity of the spin dynamics when describing the interplay of the RF irradiation with the mechanical rotation of the shift tensor. In this paper, we present a theoretical description, based on the jolting frame formalism of Caravatti et al. [J. Magn. Reson. 55, 88 (1983)], for describing the spin dynamics of a powder sample under MAS when subjected to a selective pulse of low RF-field amplitude. The formalism is used to describe the frequency stepping method under MAS, and under what circumstances the true spectrum is reproduced. We also present an experimental validation of the methodology under ultra-fast MAS with the paramagnetic materials LiMnPO4 and TbCsDPA.

Magic Angle Spinning NMR of Paramagnetic Proteins

Metal ions are ubiquitous in biochemical and cellular processes. Since many metal ions are paramagnetic due to the presence of unpaired electrons, paramagnetic molecules are an important class of targets for research in structural biology and related fields. Today, NMR spectroscopy plays a central role in the investigation of the structure and chemical properties of paramagnetic metalloproteins, linking the observed paramagnetic phenomena directly to electronic and molecular structure. A major step forward in the study of proteins by solid-state NMR came with the advent of ultrafast magic angle spinning (MAS) and the ability to use (1)H detection. Combined, these techniques have allowed investigators to observe nuclei that previously were invisible in highly paramagnetic metalloproteins. In addition, these techniques have enabled quantitative site-specific measurement of a variety of long-range paramagnetic effects. Instead of limiting solid-state NMR studies of biological systems, paramagnetism provides an information-rich phenomenon that can be exploited in these studies. This Account emphasizes state-of-the-art methods and applications of solid-state NMR in paramagnetic systems in biological chemistry. In particular, we discuss the use of ultrafast MAS and (1)H-detection in perdeuterated paramagnetic metalloproteins. Current methodology allows us to determine the structure and dynamics of metalloenzymes, and, as an example, we describe solid-state NMR studies of microcrystalline superoxide dismutase, a 32 kDa dimer. Data were acquired with remarkably short times, and these experiments required only a few milligrams of sample.

Backbone Assignment of Fully Protonated Solid Proteins by 1H Detection and Ultrafast Magic‐Angle‐Spinning NMR Spectroscopy

Narrow 1H NMR linewidths can be obtained for fully protonated protein samples in the solid state by using ultrafast magic-angle spinning (60 kHz). Medium-size microcrystalline and noncrystalline proteins can be analyzed without any need for deuteration of the protein sample. This approach provides assignments of the backbone 1H, 15N, 13Cα, and 13CO resonances and yields information about 1H–1H proximities.

Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

Combination of DQ and ZQ Coherences for Sensitive Through‐Bond NMR Correlation Experiments in Biosolids under Ultra‐Fast MAS

A double-zero quantum (DZQ)-refocused INADEQUATE experiment is introduced for J-based NMR correlations under ultra-fast (60 kHz) magic angle spinning (MAS). The experiment records two spectra in the same dataset, a double quantum-single quantum (DQ-SQ) and zero quantum-single quantum (ZQ-SQ) spectrum, whereby the corresponding signals appear at different chemical shifts in ω(1). Furthermore, the spin-state selective excitation (S(3)E) J-decoupling block is incorporated in place of the second refocusing echo of the INADEQUATE scheme, providing an additional gain in sensitivity and resolution. The two sub-spectra acquired in this way can be treated separately by a shearing transformation, producing two diagonal-free single quantum (SQ-SQ)-type spectra, which are subsequently recombined to give an additional sensitivity enhancement, thus offering an improvement greater than a factor of two as compared to the original refocused INADEQUATE experiment. The combined DZQ scheme retains transverse magnetization on the initially polarized (I) spin, which typically exhibits a longer transverse dephasing time (T(2)') than its through-bond neighbour (S). By doing so, less magnetization is lost during the refocusing periods in the sequence to give even further gains in sensitivity for the J correlations. The experiment is demonstrated for the correlation between the carbonyl (CO) and alpha (CA) carbons in a microcrystalline sample of fully protonated, [(15)N,(13)C]-labelled Cu(II),Zn(II) superoxide dismutase, and its efficiency is discussed with respect to other J-based schemes.

Nature and Structure of Aluminum Surface Sites Grafted on Silica from a Combination of High-Field Aluminum-27 Solid-State NMR Spectroscopy and First-Principles Calculations

The determination of the nature and structure of surface sites after chemical modification of large surface area oxides such as silica is a key point for many applications and challenging from a spectroscopic point of view. This has been, for instance, a long-standing problem for silica reacted with alkylaluminum compounds, a system typically studied as a model for a supported methylaluminoxane and aluminum cocatalyst. While (27)Al solid-state NMR spectroscopy would be a method of choice, it has been difficult to apply this technique because of large quadrupolar broadenings. Here, from a combined use of the highest stable field NMR instruments (17.6, 20.0, and 23.5 T) and ultrafast magic angle spinning (>60 kHz), high-quality spectra were obtained, allowing isotropic chemical shifts, quadrupolar couplings, and asymmetric parameters to be extracted. Combined with first-principles calculations, these NMR signatures were then assigned to actual structures of surface aluminum sites. For silica (here SBA-15) reacted with triethylaluminum, the surface sites are in fact mainly dinuclear Al species, grafted on the silica surface via either two terminal or two bridging siloxy ligands. Tetrahedral sites, resulting from the incorporation of Al inside the silica matrix, are also seen as minor species. No evidence for putative tri-coordinated Al atoms has been found.

Fast Resonance Assignment and Fold Determination of Human Superoxide Dismutase by High‐Resolution Proton‐Detected Solid‐State MAS NMR Spectroscopy

Re-protonation is key: A combination of a high magnetic field (1 GHz) and ultra-fast magic-angle spinning (60 kHz) allows easy detection of NMR spectra revealing details of secondary and tertiary structures of medium-sized proteins. The technique was applied to the 153-residue microcrystalline Zn II-loaded superoxide dismutase (ZnII-SOD) fully [ 2H,13C,15N]-labeled and 100% re-protonated at the exchangeable sites. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced Resolution and Coherence Lifetimes in the Solid-State NMR Spectroscopy of Perdeuterated Proteins under Ultrafast Magic-Angle Spinning

We investigate the combined effect of perdeuteration and fast magic-angle spinning on the resolution and sensitivity of proton-detected protein NMR spectra and on coherence lifetimes. With 60 kHz spinning of a microcrystalline α-spectrin SH3 sample at a field strength of 23 T, a regime is attained where there is no substantial difference in resolution between perdeuterated samples with 10 or 100% protons at the exchangeable sites.1H resolution is then limited by inhomogeneous contributions. Upon fast spinning, the most dramatic line narrowing effects are observed for residues in the loop or bend regions of the protein, probably due to the removal of destructive dynamics effects. This investigation paves the way for using samples with 100% protons at the exchangeable sites in structure determination protocols, since all backbone amide sites can now contribute to the signal.