Search

ABSTRACT In solid‐state nuclear magnetic resonance (ssNMR) spectroscopy, fast magic angle spinning (MAS) is a potent technique that efficiently reduces line broadening and makes it possible to probe structural details of biological systems in high resolution. However, its utilization in studying complex heterogeneous biomaterials such as bone in their native state has been limited. The present study has demonstrated the feasibility of acquiring two‐dimensional (2D) 1H‐1H correlation spectra for native bone using multiple‐quantum/single‐quantum correlation experiments (MQ/SQ) at fast MAS (70 kHz). This method uncovered distinct 1H–1H dipolar coupling networks involving long‐chain charged residues of collagen protein, highlighting their role in maintaining the stability of the collagen triple helix. Our study opens up new avenues for 1H‐detected multi‐quantum‐based experiments at fast MAS on native collagen‐containing biological systems to explore their complex heterogeneous structural details more efficiently. [ABSTRACT FROM AUTHOR]

Nitrogen-14 (14N) overtone (OT) spectroscopy under fast magic angle spinning (MAS) conditions (>60 kHz) has emerged as a powerful technique for observing correlations and distances between 14N and ¹H, owing to the absence of the first-order quadrupolar broadenings. In addition, 14NOT allows selective manipulation of 14N nuclei for each site. Despite extensive theoretical and experimental studies, the spin dynamics of 14NOT remains under debate. In this study, we conducted experimental investigations to assess the spin dynamics of 14NOT using the rotational-echo saturation-pulse double-resonance (RESPDOR) sequence, which monitors population transfer induced by a14NOT pulse. The 14NOT spin dynamics is well represented by a model of a two-energy-level system. Unlike spin-1/2, the maximum excitation efficiency of 14NOT coherences of powdered solids, denoted by p, depends on the radiofrequency field (rf-field) strength due to orientation dependence of effective nutation fields even when pulse lengths are optimized. It is also found that the p factor, contributing to the 14NOT spin dynamics, is nearly independent of the B0 field. Consequently, the filtering efficiency of RESPDOR experiments exhibits negligible dependence on B0 when the 14NOT pulse length is optimized. The study also identifies the optimal experimental conditions for 14NOT/¹H RESPDOR correlation experiments. [ABSTRACT FROM AUTHOR]

Nitrogen-14 (14N) overtone (OT) spectroscopy under fast magic angle spinning (MAS) conditions (>60 kHz) has emerged as a powerful technique for observing correlations and distances between 14N and 1H, owing to the absence of the first-order quadrupolar broadenings. In addition, 14NOT allows selective manipulation of 14N nuclei for each site. Despite extensive theoretical and experimental studies, the spin dynamics of 14NOT remains under debate. In this study, we conducted experimental investigations to assess the spin dynamics of 14NOT using the rotational-echo saturation-pulse double-resonance (RESPDOR) sequence, which monitors population transfer induced by a14NOT pulse. The 14NOT spin dynamics is well represented by a model of a two-energy-level system. Unlike spin-1/2, the maximum excitation efficiency of 14NOT coherences of powdered solids, denoted by p, depends on the radiofrequency field (rf-field) strength due to orientation dependence of effective nutation fields even when pulse lengths are optimized. It is also found that the p factor, contributing to the 14NOT spin dynamics, is nearly independent of the B0 field. Consequently, the filtering efficiency of RESPDOR experiments exhibits negligible dependence on B0 when the 14NOT pulse length is optimized. The study also identifies the optimal experimental conditions for 14NOT/1H RESPDOR correlation experiments.

The assignment of aromatic side-chain spins has always been more challenging than assigning backbone and aliphatic spins. Selective labeling combined with mutagenesis has been the approach for assigning aromatic spins. This manuscript reports a method for assigning aromatic spins in a fully protonated protein by connecting them to the backbone atoms using a low-power TOBSY sequence. The pulse sequence employs residual polarization and sequential acquisitions techniques to record HN- and HC-detected spectra in a single experiment. The unambiguous assignment of aromatic spins also enables the characterization of 1H–1H distance restraints involving aromatic spins. Broadband (RFDR) and selective (BASS-SD) recoupling sequences were used to generate HN-ΗC, HC-HN and HC-HC restraints involving the side-chain proton spins of aromatic residues. This approach has been demonstrated on a fully protonated U-[13C,15N] labeled GB1 sample at 95–100 kHz MAS.

This primer article introduces a practical guide for setting up 1H detection solid-state NMR experiments under fast MAS conditions for small molecules. Sample independent adjustment/calibration including magic angle adjustment, shimming, radio-frequency field calibration, Hartmann-Hahn matching adjustments, and 1H heteronuclear decoupling are demonstrated. Experimental setups which depend on samples are also shown for 1H NMR, 1H-1H homonuclear correlation measurements, and 1H-X heteronuclear correlation/distance measurements. Information obtained from each experiment is also highlighted.

Measurement of distances from dipolar couplings is essential for structural characterization, refinement and validation using the solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Particularly, knowledge about NH dipolar interactions in biological solids is important for understanding the hydrogen (H)-bonding interactions, molecular geometry and spin dynamics. In this regard, we have proposed a proton-detected two-dimensional (2D) 15N-1H dipolar coupling/1H chemical shift correlation experiment using the C-symmetry based windowless recoupling of chemical shift anisotropy (ROCSA) in combination with the DIPSHIFT pulse-based method for the measurement of short NH distances in the isotopically labeled and naturally abundant biological solids at fast magic angle spinning (MAS) rates (40–70 kHz). Our proposed method results in undistorted recoupled 15N-1H dipolar coupling powder lineshapes that are free from the recoupled 1H CSA contributions under the 15N evolution, a feature that is essential for the measurement of NH distances with improved accuracy (± 500 Hz in terms of the NH dipolar couplings). The pulse sequence developed in the present study is also insensitive to the 1H–1H homonuclear dipolar interactions, relaxation effects owing to its constant-time implementation, and t1-noise from the fluctuations in the MAS.

In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state 1H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire 13C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone 1H resonances. The performance of ten methods for recoupling of either isotropic 13C–13C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B0 field strengths (28.2 T). The concept of linearization of 13C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts.

Solid-state NMR spectroscopy is a powerful technique to study insoluble and non-crystalline proteins and protein complexes at atomic resolution. The development of proton (1H) detection at fast magic-angle spinning (MAS) has considerably increased the analytical capabilities of the technique, enabling the acquisition of 1H-detected fingerprint experiments in few hours. Here an approach based on double-quantum (DQ) 13C spectroscopy, detected on 1H, is proposed for fast MAS regime (> 60 kHz) to perform the sequential assignment of insoluble proteins of small size, without any specific deuteration requirement. By combining two three-dimensional 1H detected experiments correlating a 13C DQ dimension respectively to its intra-residue and sequential 15 N-1H pairs, a sequential walk through DQ (Ca + CO) resonance is obtained. The approach takes advantage of fast MAS to achieve an efficient sensitivity and the addition of a DQ dimension provides spectral features useful for the resonance assignment process. [ABSTRACT FROM AUTHOR]

Two-dimensional (15)N chemical shift/(1)H chemical shift and three-dimensional (1)H-(15)N dipolar coupling/(15)N chemical shift/(1)H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from (1)H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly (15)N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear (1)H-(15)N dipolar coupling frequency dimension is shown to select among (15)N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

Asparagine and glutamine side-chains can form hydrogen-bonded ladders which contribute significantly to the stability of amyloid fibrils. We show, using the example of HET-s(218–289) fibrils, that the primary amide side-chain proton resonances can be detected in cross-polarization based solid-state NMR spectra at fast magic-angle spinning (MAS). J-coupling based experiments offer the possibility to distinguish them from backbone amide groups if the spin-echo lifetimes are long enough, which turned out to be the case for the glutamine side-chains, but not for the asparagine side-chains forming asparagine ladders. We explore the sensitivity of NMR observables to asparagine ladder formation. One of the two possible asparagine ladders in HET-s(218–289), the one comprising N226 and N262, is assigned by proton-detected 3D experiments at fast MAS and significant de-shielding of one of the NH2 proton resonances indicative of hydrogen-bond formation is observed. Small rotating-frame 15N relaxation-rate constants point to rigidified asparagine side-chains in this ladder. The proton resonances are homogeneously broadened which could indicate chemical exchange, but is presently not fully understood. The second asparagine ladder (N243 and N279) in contrast remains more flexible.

Hydrogen bonds are essential for protein structure and function, making experimental access to long-range interactions between amide protons and heteroatoms invaluable. Here we show that measuring distance restraints involving backbone hydrogen atoms and carbonyl- or α-carbons enables the identification of secondary structure elements based on hydrogen bonds, provides long-range contacts and validates spectral assignments. To this end, we apply specifically tailored, proton-detected 3D (H)NCOH and (H)NCAH experiments under fast magic angle spinning (MAS) conditions to microcrystalline samples of SH3 and GB1. We observe through-space, semi-quantitative correlations between protein backbone carbon atoms and multiple amide protons, enabling us to determine hydrogen bonding patterns and thus to identify β-sheet topologies and α-helices in proteins. Our approach shows the value of fast MAS and suggests new routes in probing both secondary structure and the role of functionally-relevant protons in all targets of solid-state MAS NMR. [ABSTRACT FROM AUTHOR]

Obtaining site-specific assignments for the NMR spectra of proteins in the solid state is a significant bottleneck in deciphering their biophysics. This is primarily due to the time-intensive nature of the experiments. Additionally, the low resolution in the 1 H -dimension requires multiple complementary experiments to be recorded to lift degeneracies in assignments. We present here an approach, gleaned from the techniques used in multiple-acquisition experiments, which allows the recording of forward and backward residue-linking experiments in a single experimental block. Spectra from six additional pathways are also recovered from the same experimental block, without increasing the probe duty cycle. These experiments give intra- and inter residue connectivities for the backbone 13 C α , 15 N , 1 H N and 1 H α resonances and should alone be sufficient to assign these nuclei in proteins at MAS frequencies > 60 kHz. The validity of this approach is tested with experiments on a standard tripeptide N-formyl methionyl-leucine-phenylalanine (f-MLF) at a MAS frequency of 62.5 kHz, which is also used as a test-case for determining the sensitivity of each of the experiments. We expect this approach to have an immediate impact on the way assignments are obtained at MAS frequencies > 60 kHz . [ABSTRACT FROM AUTHOR]

We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ~50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and HN back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170–110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several HN protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra.

Chemical shift anisotropy (CSA) is a sensitive probe of electronic environment at a nucleus, and thus, it offers deeper insights into detailed structural and dynamic properties of different systems, for example, chemical, biological, and materials. Over the years, massive efforts have been made to develop recoupling methods that reintroduce CSA interaction under magic angle spinning (MAS) conditions. Most of them require slow or moderate MAS (≤20 kHz) and isotopically enriched samples. On the other hand, to the best of the authors' knowledge, no 13C or 15N CSA recoupling schemes at ultrafast MAS (≥60 kHz) suitable for cost‐effective natural abundant samples have been developed. We present here a proton‐detected 3D 15N CS/15N CSA/1H CS correlation experiment which employs 1H indirect detection for sensitivity enhancement and a γ‐encoded RNnν‐symmetry‐based CSA recoupling scheme. In particular, two different symmetries, that is, R837 and R1049, are first tested, in a 2D 15N CSA/1H CS version, on [U‐15N]‐L‐histidine·HCl·H2O as a model sample under 70 kHz MAS. Then the 3D experiment is applied on glycyl‐L‐alanine at natural abundance, resulting in site‐resolved 15N CSA lineshapes from which CSA parameters are retrieved by SIMPSON numerical fittings. We demonstrate that this 3D R‐symmetry‐based pulse sequence is highly robust with respect to wide‐range offset mismatches and weakly dependent to rf inhomogeneity within mis‐sets of ±10% from the theoretical value. [ABSTRACT FROM AUTHOR]

We report linewidth and proton T1, T1ρ and T2′ relaxation data of the model protein ubiquitin acquired at MAS frequencies up to 126 kHz. We find a predominantly linear improvement in linewidths and coherence decay times of protons with increasing spinning frequency in the range from 93 to 126 kHz. We further attempt to gain insight into the different contributions to the linewidth at fast MAS using site-specific analysis of proton relaxation parameters and present bulk relaxation times as a function of the MAS frequency. For microcrystalline fully-protonated ubiquitin, inhomogeneous contributions are only a minor part of the proton linewidth, and at 126 kHz MAS coherent effects are still dominating. We furthermore present site-specific proton relaxation rate constants during a spinlock at 126 kHz MAS, as well as MAS-dependent bulk T1ρ (1HN). [ABSTRACT FROM AUTHOR]

We analyze the performances and the optimization of 1 H-{ I } HMQC experiments using basic and interleaved DANTE schemes for the indirect detection of nuclei I  = 1/2 or 1 exhibiting wide lines dominated by chemical shift anisotropy (CSA) or quadrupole interaction, respectively. These sequences are first described using average Hamiltonian theory. Then, we analyze using numerical simulations (i) the optimal lengths of the DANTE train and the individual pulses, (ii) the robustness of these experiments to offset, and (iii) the optimal choice of the defocusing and refocusing times for both 1 H-{ I } J - and D -HMQC sequences for 195 Pt ( I  = 1/2) and 14 N ( I  = 1) nuclei subject to large CSA and quadrupole interaction, respectively. These simulations are compared to 1 H-{ 14 N} D -HMQC experiments on γ-glycine and L-histidine.HCl at B 0  = 18.8 T and MAS frequency of 62.5 kHz. The present study shows that (i) the optimal defocusing and refocusing times do not depend on the chosen DANTE scheme, (ii) the DANTE trains must be applied with the highest rf-field compatible with the probe specifications and the stability of the sample, (iii) the excitation bandwidth along the indirect dimension of HMQC sequence using DANTE trains is inversely proportional to their length, (iv) interleaved DANTE trains increase the excitation bandwidth of these sequences, and (v) dephasing under residual 1 H– 1 H and 1 H- I dipolar couplings, as well as 14 N second-order quadrupole interaction, during the length of the DANTE scheme attenuate the transfer efficiency. [ABSTRACT FROM AUTHOR]

The application of overtone nuclear magnetic resonance (OT NMR) to symmetric spin transitions of integer quadrupolar nuclei is of considerable interest since this transition is immune to the first-order quadrupolar interaction, thus resulting in narrow NMR lines. Owing to its roles in nature and its high natural abundance, 14 N ( I  = 1) OT NMR has been explored, in which the indirect and/or direct acquisitions of 14 N OT were experimentally demonstrated. However, other than 14 N nucleus, no OT NMR observation of other integer quadrupolar nuclei has been reported in the literature. In this work, we extend the application of OT NMR to another integer quadrupolar nucleus, namely 10 B ( I =  3). However, this is not straightforward owing to the unfavorable characteristics of 10 B isotope. Here, for the first time, we present the selective acquisition of 10 B central (−1 ↔ +1) OT NMR via detection of 1 H nuclei on perborate monohydrate sample. Numerical calculations are in a good agreement with the experimental results. Both show that the optimal sensitivity is achieved when the carrier frequency is applied at the second OT spinning sideband, i.e. an offset of twice of the spinning frequency from the center band. [ABSTRACT FROM AUTHOR]

Chemical characterisation of active pharmaceutical compounds can be challenging, especially when these molecules exhibit tautomeric or desmotropic behaviour. The complexity can increase manyfold if these molecules are not susceptible to crystallisation. Solid-state NMR has been employed effectively for characterising such molecules. However, characterisation of a molecule is just a first step in identifying the differences in the crystalline structure. 1 H solid-state Nuclear Magnetic Resonance (ssNMR) studies on these molecules at fast magic-angle-spinning frequencies can provide a wealth of information and may be used along with ab initio calculations to predict the crystal structure in the absence of X-ray crystallographic studies. In this work, we attempted to use solid-state NMR to measure 1 H - 1 H distances that can be used as restraints for crystal structure calculations. We performed studies on the desmotropic forms of albendazole.

Through-bond J -coupling based experiments in solid-state NMR spectroscopy are challenging because the J couplings are typically much smaller than the dipolar couplings. This often leads to a lower transfer efficiency compared to dipolar-coupling based sequences. One of the reasons for the low transfer efficiency are the second-order cross terms involving the strong heteronuclear dipolar couplings leading to fast magnetization decay. Here, we show that by employing a symmetry-based C9 sequence, which was carefully selected to suppress second-order terms, efficient polarization transfers of up to 80% can be achieved without decoupling on fully protonated two-spin model systems at a MAS frequency of 55.5 kHz with rf-field amplitudes of about 25 kHz. In addition, we analyse the effects of rf inhomogeneity and crystallites selection due to the polarization preparation method on the TOBSY transfer efficiency. We demonstrate on small model substances as well as on deuterated and 100% back-exchanged ubiquitin that C 9 39 1 and C 9 48 1 are efficient and practical TOBSY sequences at experimental conditions ranging from proton Larmor frequencies of 400–850 MHz, and MAS frequencies ranging from 55.5 to 111.1 kHz. [ABSTRACT FROM AUTHOR]

Fast magic angle spinning (MAS) and proton detection has found widespread application to enhance the sensitivity of solid-state NMR experiments with spin-1/2 nuclei such as 13 C, 15 N and 29 Si, however, this approach is not yet routinely applied to half-integer quadrupolar nuclei. Here we have investigated the feasibility of using fast MAS and proton detection to enhance the sensitivity of solid-state NMR experiments with half-integer quadrupolar nuclei. The previously described dipolar hetero-nuclear multiple quantum correlation (D-HMQC) and dipolar refocused insensitive nuclei enhanced by polarization transfer (D-RINEPT) pulse sequences were used for proton detection of half-integer quadrupolar nuclei. Quantitative comparisons of signal-to-noise ratios and the sensitivity of proton detected D-HMQC and D-RINEPT and direct detection spin echo and quadrupolar Carr-Purcell Meiboom-Gill (QCPMG) solid-state NMR spectra, demonstrate that one dimensional proton detected experiments can provide sensitivity similar to or exceeding that obtainable with direct detection QCPMG experiments. 2D D-HMQC and D-RINEPT experiments provide less sensitivity than QCPMG experiments but proton detected 2D hetero-nuclear correlation solid-state NMR spectra of half-integer nuclei can still be acquired in about the same time as a 1D spin echo spectrum. Notably, the rarely used D-RINEPT pulse sequence is found to provide similar, or better sensitivity than D-HMQC in some cases. Proton detected D-RINEPT benefits from the short longitudinal relaxation times ( T 1 ) normally associated with half-integer quadrupolar nuclei, it can be combined with existing signal enhancement methods for quadrupolar nuclei, and t 1 -noise in the indirect dimension can easily be removed by pre-saturation of the 1 H nuclei. The rapid acquisition of proton detected 2D HETCOR solid-state NMR spectra of a range of half-integer quadrupolar nuclei such as 17 O, 27 Al, 35 Cl and 71 Ga is demonstrated. [ABSTRACT FROM AUTHOR]

The mutual orientation of nuclear spin interaction tensors provides critical information on the conformation and arrangement of molecules in chemicals, materials, and biological systems at an atomic level. Proton is a ubiquitous and important element in a variety of substances, and its NMR is highly sensitive due to their virtually 100% natural abundance and large gyromagnetic ratio. Nevertheless, the measurement of mutual orientation between the 1H CSA tensors has remained largely untouched in the past due to strong 1H–1H homonuclear interactions in a dense network of protons. In this study, we have developed a proton-detected 3D 1H CSA/1H CSA/1H CS correlation method that utilizes three techniques to manage homonuclear interactions, namely fast magic-angle spinning, windowless C -symmetry-based CSA recoupling (windowless-ROCSA), and a band-selective 1H–1H polarization transfer. The asymmetric 1H CSA/1H CSA correlated powder patterns produced by the C-symmetry-based methods are highly sensitive to the sign and asymmetry parameter of the 1H CSA, and the Euler angle β as compared to the symmetric pattern obtained by the existing γ -encoded R -symmetry-based CSA/CSA correlation methods and allows a larger spectral area for data fitting. These features are beneficial for determining the mutual orientation between the nuclear spin interaction tensors with improved accuracy. [Display omitted] • A 3D 1H CSA/1H CSA/1H CS sequence with the windowless ROCSA is developed for measuring mutual orientation of CSA tensors. • 2D 1H CSA/1H CSA powder pattern is sensitive to the sign of the 1H CSA and all the Euler angles α , β , and γ. • The sequence can be applied to strongly dipolar coupled protons. [ABSTRACT FROM AUTHOR]

[Display omitted] • Proton-detected 3D 15N-1H dipolar coupling/1H CSA/1H CS pulse sequence is developed at fast MAS. • Relative orientation between the 15N-1H dipolar coupling and 1H CSA tensors is measured. • Windowless C-symmetry based ROCSA-DIPSHIFT is integrated with ROCSA for the recoupling of the 15N-1H dipolar coupling and 1H CSA tensors, respectively. • The 2D 15N-1H dipolar coupling/1H CSA powder pattern is sensitive to the sign and asymmetry of the 1H CSA, and exhibits profound dependence on the dipolar PAS angles. In this work, we have proposed a proton-detected three-dimensional (3D) 15N-1H dipolar coupling (DIP)/1H chemical shift anisotropy (CSA)/1H chemical shift (CS) correlation experiment to measure the relative orientation between the 15N-1H dipolar coupling and the 1H CSA tensors under fast magic angle spinning (MAS) solid-state NMR. In the 3D correlation experiment, the 15N-1H dipolar coupling and 1H CSA tensors are recoupled using our recently developed windowless C -symmetry-based C 3 3 1 -ROCSA (recoupling of chemical shift anisotropy) DIPSHIFT and C 3 3 1 -ROCSA pulse-based methods, respectively. The 2D 15N-1H DIP/1H CSA powder lineshapes extracted using the proposed 3D correlation method are shown to be sensitive to the sign and asymmetry of the 1H CSA tensor, a feature that allows the determination of the relative orientation between the two correlating tensors with improved accuracy. The experimental method developed in this study is demonstrated on a powdered U-15N L-Histidine.HCl·H 2 O sample. [ABSTRACT FROM AUTHOR]

[Display omitted] • T -HMQC filter is applied to suppress 1H resonances except for NH protons. • T -HMQC filtered PM-S-RESPDOR measures accurate 1H-14N distances. • 1H peaks overlapped to NH protons are eliminated before PM-S-RESPDOR. 1H-14N internuclear distances are readily and accurately measured using the symmetry-based phase-modulated resonance-echo saturation-pulse double-resonance (PM-S-RESPDOR) method in rigid solids. The fraction curve, (S 0 – S')/S 0 , is represented by a single variable of a 1H-14N heteronuclear dipolar coupling, where S 0 and S' are the PM-S-RESPDOR signal intensity with and without 14N PM saturation pulse, respectively. Analytical equation of the fraction curve easily provides 1H-14N couplings. This treatment is only applicable when NH proton resonance is well separated from the other proton peaks. With the limited 1H resolution even at fast MAS > 60 kHz, unfortunately, this condition is not necessarily satisfied especially in multi-component systems which often appear in pharmaceutical applications. To overcome this problem, T -HMQC filtering is applied to suppress the 1H signals other than NH proton prior to the PM-S-RESPDOR experiments. The method is well demonstrated on two components acetaminophen-oxalic acid (APAP-OXA) systems. Further analysis of orientation dependence of T -HMQC and PM-S-RESPDOR shows that the analytical equation can be safely applied in the analysis of T -HMQC filtered PM-S-RESPDOR experiments. [ABSTRACT FROM AUTHOR]

Fast magic-angle spinning (>60 kHz) has many advantages but makes spin-diffusion-type proton-proton long-range polarization transfer inefficient and highly dependent on chemical-shift offset. Using 100%-HN-[H,C,N]-ubiquitin as a model substance, we quantify the influence of the chemical-shift difference on the spin diffusion between proton spins and compare two experiments which lead to an improved chemical-shift compensation of the transfer: rotating-frame spin diffusion and a new experiment, reverse amplitude-modulated MIRROR. Both approaches enable broadband spin diffusion, but the application of the first variant is limited due to fast spin relaxation in the rotating frame. The reverse MIRROR experiment, in contrast, is a promising candidate for the determination of structurally relevant distance restraints. The applied tailored rf-irradiation schemes allow full control over the range of recoupled chemical shifts and efficiently drive spin diffusion. Here, the relevant relaxation time is the larger longitudinal relaxation time, which leads to a higher signal-to-noise ratio in the spectra. [ABSTRACT FROM AUTHOR]

The performance of heteronuclear spin decoupling sequences in solid-state NMR severely degrades when the proton radiofrequency (RF) nutation frequencies ( ν 1 ) are close to or at multiples of magic-angle spinning (MAS) frequency ( ν r ) that are referred to as rotary-resonance recoupling conditions ( ν 1 = n · ν r ). Recently, two schemes, namely, PISSARRO and rCW ApA , have been shown to be less affected by the problem of MAS and RF interference, specifically at the n = 2 rotary-resonance recoupling condition, especially in the fast MAS regime. Here, we systematically evaluate the loss in intensity of several heteronuclear spin decoupling sequences at the n = 1, 2 conditions compared to high-power decoupling in the fast-MAS regime. We propose that in the fast-MAS regime (above 40 kHz) the entire discussion about RF and MAS interference can be avoided by using appropriate low-power decoupling sequences which give comparable performance to decoupling sequences with high-power 1 H irradiation of ca.195 kHz. [ABSTRACT FROM AUTHOR]

In spite of tremendous progress made in pulse sequence designs and sophisticated hardware developments, methods to improve sensitivity and resolution in solid-state NMR (ssNMR) are still emerging. The rate at which sample is spun at magic angle determines the extent to which sensitivity and resolution of NMR spectra are improved. To this end, the prime objective of this article is to give a comprehensive theoretical and experimental framework of fast magic angle spinning (MAS) technique. The engineering design of fast MAS rotors based on spinning rate, sample volume, and sensitivity is presented in detail. Besides, the benefits of fast MAS citing the recent progress in methodology, especially for natural abundance samples are also highlighted. The effect of the MAS rate on 1 H resolution, which is a key to the success of the 1 H inverse detection methods, is described by a simple mathematical factor named as the homogeneity factor k . A comparison between various 1 H inverse detection methods is also presented. Moreover, methods to reduce the number of spinning sidebands (SSBs) for the systems with huge anisotropies in combination with 1 H inverse detection at fast MAS are discussed. [ABSTRACT FROM AUTHOR]

We show that multidimensional solid-state NMR 13C–13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule. [Display omitted] • Unprecedented sensitivity using recently developed AsymPol-POK hyperpolarizing agent. • Carbon-carbon correlation experiments at natural abundance on 1–2 mg of sample. • Examples on amyloid fibrils and microcrystals of pharmaceuticals at natural isotopic abundance. [ABSTRACT FROM AUTHOR]

[Display omitted] • Exhausting 1H polarization in each transient scan to enhance NMR sensitivity. • Several new sequences that hybrid HOMCOR and HETCOR sequences are proposed. • One experiment to obtain 3D 1H HOMCOR and 2D 1H-X HETCOR simultaneously. • One experiment to obtain 2D 1H DQ/SQ, SQ/SQ and 2D 1H-X HETCOR simultaneously. Proton-detected solid-state NMR spectroscopy is emerging as a unique tool for atomic characterization of organic solids due to the boost of resolution and sensitivity afforded by the combined use of high magnetic field and ultrafast magic angle spinning (MAS). Here, we proposed a new set of proton-detected solid-state NMR sequences that hybrid multi-dimensional 1H–1H homonuclear chemical shift correlation (HOMCOR) and two-dimensional 1H–13C heteronuclear chemical shift correlation (HETCOR) sequences into a single experiment, enabling the simultaneous acquisition of multidimensional HOMCOR and HETCOR spectra and thus significant time savings. Based on the core idea of exhausting 1H polarization in each transient scan, we firstly demonstrated that 3D 1H multiple-quantum (MQ) HOMCOR sequence can be combined with 2D HETCOR sequence into a single experiment, leading to the simultaneous acquisition of a 3D 1H MQ HOMCOR and a 2D 1H–13C HETCOR spectrum. Besides, we also showed that 2D 1H/1H double-quantum/single-quantum (DQ/SQ) and single-quantum/single-quantum (SQ/SQ) HOMCOR sequence can be simultaneously combined with HETCOR sequence either, and thus three spectra can be simultaneously obtained from one experiment, including 2D 1H DQ/SQ, 2D 1H SQ/SQ and 2D 1H–13C HETCOR spectra. Since there is only one recycle delay in each experiment, experimental time is substantially reduced compared to separate acquisition of each multi-dimensional solid-state NMR spectrum. Furthermore, those new sequences can be implemented on any standard solid-state spectrometer with only one receiver. Thus, we foresee that these approaches can be valuable for the study of a broad range of molecular systems, including polymers, pharmaceuticals, covalent-organic frameworks (COF) and so on. [ABSTRACT FROM AUTHOR]

This study demonstrates a novel spectral editing technique for protein solid-state NMR (SSNMR) to simplify the spectrum drastically and to reduce the ambiguity for protein main-chain signal assignments in fast magic-angle-spinning (MAS) conditions at a wide frequency range of 40-80 kHz. The approach termed HIGHLIGHT (Wang et al., in Chem Comm 51:15055-15058, ) combines the reverse C, N-isotope labeling strategy and selective signal quenching using the frequency-selective REDOR pulse sequence under fast MAS. The scheme allows one to selectively observe the signals of 'highlighted' labeled amino-acid residues that precede or follow unlabeled residues through selectively quenching CO or N signals for a pair of consecutively labeled residues by recoupling CO-N dipolar couplings. Our numerical simulation results showed that the scheme yielded only ~15 % loss of signals for the highlighted residues while quenching as much as ~90 % of signals for non-highlighted residues. For lysine-reverse-labeled micro-crystalline GB1 protein, the 2D N/C correlation and 2D C/CO correlation SSNMR spectra by the HIGHLIGHT approach yielded signals only for six residues following and preceding the unlabeled lysine residues, respectively. The experimental dephasing curves agreed reasonably well with the corresponding simulation results for highlighted and quenched residues at spinning speeds of 40 and 60 kHz. The compatibility of the HIGHLIGHT approach with fast MAS allows for sensitivity enhancement by paramagnetic assisted data collection (PACC) and H detection. We also discuss how the HIGHLIGHT approach facilitates signal assignments using C-detected 3D SSNMR by demonstrating full sequential assignments of lysine-reverse-labeled micro-crystalline GB1 protein (~300 nmol), for which data collection required only 11 h. The HIGHLIGHT approach offers valuable means of signal assignments especially for larger proteins through reducing the number of resonance and clarifying multiple starting points in sequential assignment with enhanced sensitivity. [ABSTRACT FROM AUTHOR]

A selective saturation pulse at fast magic angle spinning (MAS) frequencies (60+ kHz) suppresses t 1 noise in the indirect dimension of two-dimensional 1 H MAS NMR spectra. The method is applied to a synthetic nucleoside with an intense methyl 1 H signal due to triisopropylsilyl (TIPS) protecting groups. Enhanced performance in terms of suppressing the methyl signal while minimising the loss of signal intensity of nearby resonances of interest relies on reducing spin diffusion – this is quantified by comparing two-dimensional 1 H NOESY-like spin diffusion spectra recorded at 30–70 kHz MAS. For a saturation pulse centred at the methyl resonance, the effect of changing the nutation frequency at different MAS frequencies as well as the effect of changing the pulse duration is investigated. By applying a pulse of duration 30 ms and nutation frequency 725 Hz at 70 kHz MAS, a good compromise of significant suppression of the methyl resonance combined with the signal intensity of resonances greater than 5 ppm away from the methyl resonance being largely unaffected is achieved. The effectiveness of using a selective saturation pulse is demonstrated for both homonuclear 1 H– 1 H double quantum (DQ)/single quantum (SQ) MAS and 14 N– 1 H heteronuclear multiple quantum coherence (HMQC) two-dimensional solid-state NMR experiments. [ABSTRACT FROM AUTHOR]

Fast magic angle spinning (MAS) NMR spectroscopy is emerging as an essential analytical and structural biology technique. Large resolution and sensitivity enhancements observed under fast MAS conditions enable structural and dynamics analysis of challenging systems, such as large macromolecular assemblies and isotopically dilute samples, using only a fraction of material required for conventional experiments. Homonuclear dipolar-based correlation spectroscopy constitutes a centerpiece in the MAS NMR methodological toolbox, and is used essentially in every biological and organic system for deriving resonance assignments and distance restraints information necessary for structural analysis. Under fast MAS conditions (rotation frequencies above 35-40 kHz), dipolar-based techniques that yield multi-bond correlations and non-trivial distance information are ineffective and suffer from low polarization transfer efficiency. To overcome this limitation, we have developed a family of experiments, CORD-RFDR. These experiments exploit the advantages of both zero-quantum RFDR and spin-diffusion based CORD methods, and exhibit highly efficient and broadband dipolar recoupling across the entire spectrum, for both short-range and long-range correlations. We have verified the performance of the CORD-RFDR sequences experimentally on a U-C,N-MLF tripeptide and by numerical simulations. We demonstrate applications of 2D CORD-RFDR correlation spectroscopy in dynein light chain LC8 and HIV-1 CA tubular assemblies. In the CORD-RFDR spectra of LC8 acquired at the MAS frequency of 40 kHz, many new intra- and inter-residue correlations are detected, which were not observed with conventional dipolar recoupling sequences. At a moderate MAS frequency of 14 kHz, the CORD-RFDR experiment exhibits excellent performance as well, as demonstrated in the HIV-1 CA tubular assemblies. Taken together, the results indicate that CORD-RFDR experiment is beneficial in a broad range of conditions, including both high and moderate MAS frequencies and magnetic fields. [ABSTRACT FROM AUTHOR]

Heteronucleus-detected dipolar based correlation spectroscopy is established for assignments of H, C, and N resonances and structural analysis in fully protonated proteins. We demonstrate that C detected 3D experiments are highly efficient and permit assignments of the majority of backbone resonances, as shown in an 89-residue dynein light chain 8, LC8 protein. With these experiments, we have resolved many ambiguities that were persistent in our previous studies using moderate MAS frequencies and lacking the H dimension. The availability of H isotropic chemical shifts measured with the heteronucleus-detected fast-MAS experiments presented here is essential for the accurate determination of the H CSA tensors, which provide very useful structural probe. Finally, our results indicate that C detection in fast-MAS HETCOR experiments may be advantageous compared with H detection as it yields datasets of significantly higher resolution in the C dimension than the H detected HETCOR versions. [ABSTRACT FROM AUTHOR]

Solid-state Magnetic Resonance has been greatly developed over the past decade. Higher field spectrometers and other technical developments are expected to lead to further significant improvements.

Analysis of short-to-intermediate range intermolecular interactions offers a great way of characterizing the solid-state organization of small molecules and materials. This can be achieved by two-dimensional (2D) homo- and heteronuclear correlation NMR spectroscopy, for example, by carrying out experiments at high magnetic fields in conjunction with fast magic-angle spinning (MAS) techniques. But, detecting 2D peaks for heteronuclear dipolar coupled spin pairs separated by greater than 3 Å is not always straightforward, particularly when low-gamma quadrupolar nuclei are involved. Here, we present a 2D correlation NMR experiment that combines the advantages of heteronuclear-multiple quantum coherence (HMQC) and proton-based spin-diffusion (SD) pulse sequences using radio-frequency-driven-recouping (RFDR) to probe inter and intramolecular 1H-X (X = 14N, 35Cl) interactions. This experiment can be used to acquire 2D 1H{X}-HMQC filtered 1H–1H correlation as well as 2D 1H-X HMQC spectra. Powder forms of dopamine·HCl and l -histidine·HCl·H 2 O are characterized at high fields (21.1 T and 18.8 T) with fast MAS (60 kHz) using the 2D HMQC-SD-RFDR approach. Solid-state NMR results are complemented with NMR crystallography analyses using the gauge-including projector augmented wave (GIPAW) approach. For histidine·HCl·H 2 O, 2D peaks associated with 14N–1H–1H and 35Cl–1H–1H distances of up to 4.4 and 3.9 Å have been detected. This is further corroborated by the observation of 2D peaks corresponding to 14N–1H–1H and 35Cl–1H–1H distances of up to 4.2 and 3.7 Å in dopamine·HCl, indicating the suitability of the HMQC-SD-RFDR experiments for detecting medium-range proximities in molecular solids. [Display omitted] • HMQC-SD-RFDR experiment is presented to analyze solid-state packing interactions. • Relayed 14N–1H–1H and 35Cl–1H–1H proximities are detected in dopamine·HCl. • Experiments are carried out at high fields (21.1 T and 18.8 T) with 60 kHz MAS. • NMR crystallography analysis is presented for dopamine·HCl. [ABSTRACT FROM AUTHOR]

[Display omitted] • Bimodal Floquet theory is used to understand the mechanism of site-directed homonuclear polarization exchange during BASS-SD recoupling. • BASS-SD mediates correlations between only chemically similar protons. • A new offset-tuned BASS-SD recoupling condition provides spatial correlation between both chemically similar and chemically different proton spins simultaneously (HN-Hα and HN– HN). • Sim-CP, co-evolution, simultaneous and sequential acquisition combined with offset-tuned BASS-SD allows observation of HN-HN, Hα-Hα, HN-Hα and Hα-HN restraints for proteins in one experiment at fast-MAS. Band Selective Spectral Spin-Diffusion (BASS-SD) is a method to obtain selective 1H–1H contacts between chemically similar protons within a distance range of 5–6 Å in fully protonated proteins. BASS-SD combines low-amplitude proton spinlock radio frequency (rf) pulses with fast MAS frequency to enable selective polarization exchange in fully protonated molecules. The selectivity of transfer is dictated by the bandwidth of the spinlock pulse and has been used to observe selective HN-HN, Hα-Ηα and Hmethyl-Hmethyl correlations. These proton-proton spatial contacts are similar to those observed in perdeuterated samples and serve as useful structural restraints towards de novo protein structure determination. This study employs bimodal Floquet theory to derive the first- and second-order effective Hamiltonians necessary to understand the spin dynamics during BASS-SD. Analytical calculations combined with numerical simulations delineate two different mechanisms for polarization transfer amongst the proton spins. The BASS-SD recoupling condition has been reoptimized to observe selective correlations between chemically different protons (e.g., HN-Hα) while retaining the spatial contacts between chemically similar protons (e.g., HN-HN). The new BASS-SD condition is integrated with simultaneous and sequential acquisition approaches to generate four different types of structural restraints (HN-HN, Hα-Ηα, HN-Hα, Hα-HN) in one experiment. The approach has been demonstrated on microcrystalline U-[13C,15N] labeled GB1 protein at ∼ 95–100 kHz MAS. [ABSTRACT FROM AUTHOR]

Abstract: Two-dimensional indirectly detected through-space and through-bond 1H{15N} solid-state NMR experiments utilizing fast magic angle spinning (MAS) and homonuclear multipulse 1H decoupling are evaluated. Remarkable efficiency of polarization transfer can be achieved at a MAS rate of 40kHz by both cross-polarization and INEPT, which makes these methods applicable for routine characterizations of natural abundance solids. The first measurement of 2D 1H{15N} HETCOR spectrum of natural abundance surface species is also reported. [Copyright &y& Elsevier]

Abstract: We explore modulation-sideband recoupling conditions of the 13C–13C Second-order Hamiltonian among Analogous nuclei plus pulse sequence (SHA+), and found that this sequence can be used in two different recoupling regimes. The first regime, ν R > , is recommended for broad-band recoupling to avoid any rotational resonance broadening. In this regime, the spinning speed should be only slightly larger than , to obtain the best transfer efficiency. The second regime, ν R < , can be used to observe long-range constraints with lower spinning speed, which increases the transfer efficiency, and may allow using bigger rotors to increase the S/N ratio. [Copyright &y& Elsevier]

Highlights: [•] We present COmbined -Driven (CORD) sequences for spin diffusion at fast MAS (νr ⩾40kHz). [•] We show that CORD sequences exhibit broadband homonuclear dipolar recoupling. [•] CORD sequences display uniform cross peak intensities across the entire spectrum. [•] CORD sequences are advantageous for homonuclear spectroscopy in biological solids. [ABSTRACT FROM AUTHOR]

Abstract: A simple method is proposed for separating NMR resonances from protonated and non-protonated aromatic carbons in solids under fast magic angle spinning (MAS). The approach uses a MAS-synchronized spin-echo to exploit the differences in rotational recoupling of the dipolar interactions while fully refocusing the isotropic chemical shifts. This strategy extends the relevant time scale of spin evolution to milliseconds and circumvents the limitation of the traditional dipolar dephasing method, which in fast rotating solids is disrupted by rotational refocusing. The proposed approach can be used for quantitative measurement of carbon aromaticities in complex solids with poorly resolved spectra, as demonstrated for model compounds. [Copyright &y& Elsevier]

Abstract: The use of mixed surfactants in the synthesis of mesoporous silica nanoparticles (MSNs) is of importance in the context of adjusting pore structures, sizes and morphologies. In the present study, the arrangement of molecules in micelles produced from a mixture of two surfactants, cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB) was detailed by solid-state NMR spectroscopy. Proximities of methyl protons in the trimethylammonium headgroup of CTAB and protons in the pyridinium headgroup of CPB were observed under fast magic angle spinning (MAS) by 1H−1H double quantum (DQ) MAS NMR and NOESY. This result suggested that CTAB and CPB co-exist in the pores without forming significant monocomponent domain structures. 1H−29Si heteronuclear correlation (HETCOR) NMR showed that protons in the headgroups of CTAB are in closer proximity to the silica surface than those in the CPB headgroups. The structural information obtained in this investigation leads to better understanding of the mechanisms of self-assembly and their role in determining the structure and morphology of mesoporous materials. [Copyright &y& Elsevier]

Abstract: A simple method is shown for optimization of 1H homonuclear dipolar decoupling at MAS rates exceeding 10kHz. By monitoring the intensity of a spin-echo under the decoupling conditions, it is possible to optimize the amplitude of the RF magnetic field, the cycle time of the decoupling sequence and the resonance offset within minutes. As a result, the decoupling efficiency can be quickly and reliably fine-tuned without using a reference sample. The utility of this method has been confirmed by studying the resolution patterns for the supercycled PMLG scheme, which were found to be in excellent agreement with earlier theoretical predictions and verified in high-resolution 2D 1H–1H experiments. [Copyright &y& Elsevier]