(15)N-(1)H bond length determination in natural abundance by inverse detection in fast-MAS solid-state NMR spectroscopy

A solid-state 15N-1H correlation NMR experiment is presented, which provides a substantial gain in signal sensitivity by 1H inverse detection under fast MAS conditions and allows for the precise determination of NH bond lengths via heteronuclear 1H-15N dipole-dipole couplings on samples naturally abundant in 15N. Pulsed-field gradients or, alternatively, radio frequency pulses ensure suppression of unwanted 1H signals. In this way, natural-abundance 15N-1H correlation NMR spectroscopy becomes feasible in the solid state with experiment times of a few hours. The dipole-dipole coupling constants are extracted from spinning sideband patterns generated by recently developed recoupling strategies. The information on 15N/1H chemical shifts and quantitative 15N-1H couplings can readily be combined in a single two-dimensional spectrum using a split-t1 approach.     

Sensitive high resolution inverse detection NMR spectroscopy of proteins in the solid state

A new indirect detection scheme for obtaining (15)N/(1)H shift correlation spectra in crystalline proteins is described. Excellent water suppression is achieved without the need for pulsed field gradients, and using only a 2-step phase cycle. Careful attention to overall NMR instrument stability was found critical for obtaining the best resolution and sensitivity. Magnetic dilution by deuteration of the protein in combination with high-speed magic angle spinning produces (1)H resonances averaging only 0.22 ppm in width, and in some cases lines as narrow as 0.17 ppm are obtained. In application to two different polymorphs of ubiquitin, structure dependent differences in both (15)N and (1)H amide chemical shifts are observed. In one case, distinct shifts for different molecules in the asymmetric unit are seen, and all differ substantially from solution NMR shifts. A gain of 7 in sensitivity makes the method competitive with solution NMR as long as nanocrystalline samples are available.     

Proton-detected solid-state NMR spectroscopy of fully protonated proteins at 40 kHz magic-angle spinning

Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or mass-limited systems requires further improvements in spectral sensitivity, for which inverse detection of 13C and 15N signals with 1H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with approximately 30 kHz magic-angle spinning (MAS). Here we focus on experimental schemes for proteins with approximately 100% protonation. Full protonation simplifies sample preparation and permits more complete chemical shift information to be obtained from a single sample. We demonstrate experimental schemes using the fully protonated, uniformly 13C,15N-labeled protein GB1 at 40 kHz MAS rate with 1.6-mm rotors. At 500 MHz proton frequency, 1-ppm proton line widths were observed (500 +/- 150 Hz), and the sensitivity was enhanced by 3 and 4 times, respectively, versus direct 13C and 15N detection. The enhanced sensitivity enabled a family of 3D experiments for spectral assignment to be performed in a time-efficient manner with less than a micromole of protein. CANH, CONH, and NCAH 3D spectra provided sufficient resolution and sensitivity to make full backbone and partial side-chain proton assignments. At 750 MHz proton frequency and 40 kHz MAS rate, proton line widths improve further in an absolute sense (360 +/- 115 Hz). Sensitivity and resolution increase in a better than linear manner with increasing magnetic field, resulting in 14 times greater sensitivity for 1H detection relative to that of 15N detection.     

High resolution 1H detected 1H,13C correlation spectra in MAS solid-state NMR using deuterated proteins with selective 1H,2H isotopic labeling of methyl groups

MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme allows the acquisition of proton carbon correlation spectra with a resolution comparable to that in solution-state NMR experiments. We observe an increase in resolution by a factor of 10-15 compared to standard heteronuclear correlation experiments using PMLG for 1H,1H dipolar decoupling in the indirect dimension. At the same time, the full sensitivity of the proton-based experiment is retained. In comparison to the heteronuclear detected version of the experiment, a gain in sensitivity of a factor of approximately 4.7 is achieved.     

High resolution 1H-detected solid-state NMR spectroscopy of protein aliphatic resonances: access to tertiary structure information

Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely (13)C,(13)C distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-(13)C glycerol or [2]-(13)C glycerol to dilute the (13)C spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-γ nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the (13)C or (15)N detected version of the experiment. We report here a new labeling scheme, which enables (1)H-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken α-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic (1)H resonances. We show further that (13)C resolved 3D-(1)H,(1)H correlation experiments yield access to long-range proton-proton distances in the protein.     

1H‐19F REDOR‐filtered NMR spin diffusion measurements of domain size in heterogeneous polymers

Solid state NMR spectroscopy is inherently sensitive to chemical structure and composition and thus makes an ideal method to probe the heterogeneity of multicomponent polymers. Specifically, NMR spin diffusion experiments can be used to extract reliable information about spatial domain sizes on multiple length scales, provided that magnetization selection of one domain can be achieved. In this paper, we demonstrate the preferential filtering of protons in fluorinated domains during NMR spin diffusion experiments using ¹H‐¹⁹F heteronuclear dipolar dephasing based on rotational echo double resonance (REDOR) MAS NMR techniques. Three pulse sequence variations are demonstrated based on the different nuclei detected: direct ¹H detection, plus both ¹H?¹³C cross polarization and ¹H?¹⁹F cross polarization detection schemes. This ¹H‐¹⁹F REDOR‐filtered spin diffusion method was used to measure fluorinated domain sizes for a complex polymer blend. The efficacy of the REDOR‐based spin filter does not rely on spin relaxation behavior or chemical shift differences and thus is applicable for performing NMR spin diffusion experiments in samples where traditional magnetization filters may prove unsuccessful. This REDOR‐filtered NMR spin diffusion method can also be extended to other samples where a heteronuclear spin pair exists that is unique to the domain of interest.     

Enhanced sensitivity and resolution in (1)H solid-state NMR spectroscopy of paramagnetic complexes under very fast magic angle spinning

High-resolution NMR spectroscopy for paramagnetic complexes in solids has been rarely performed because of its limited sensitivity and resolution due to large paramagnetic shifts and associated technical difficulties. The present study demonstrates that magic angle spinning (MAS) at speeds exceeding 20 kHz provides unusually high sensitivity and excellent resolution in 1H solid-state NMR (SSNMR) for paramagnetic systems. Spinning-speed dependence of 1H MAS spectra showed that very fast MAS (VFMAS) at 24-28 kHz enhanced sensitivity by a factor of 12-18, compared with the sensitivity of 1H SSNMR spectra under moderate MAS at 10 kHz, for Cu(dl-alanine)2.H2O and Mn(acac)3, for which the spectral ranges due to 1H paramagnetic shifts reach 200 and 1000 ppm, respectively. It was theoretically and experimentally confirmed that the absolute sensitivity of 1H VFMAS for small paramagnetic complexes such as Cu(dl-alanine)2 can be an order of magnitude higher than that of equimolar diamagnetic ligands because of short 1H T1 ( approximately 1 ms) of the paramagnetic systems and improved sensitivity under VFMAS. On the basis of this demonstrated high sensitivity, 1H SSNMR micro analysis of paramagnetic systems in a nanomole scale is proposed. Applications were performed on two polymorphs of Cu(II)(8-quinolinol)2, which is a suppressor of human cancer cells. It was demonstrated that 1H VFMAS SSNMR spectra accumulated for 20 nmol of the polycrystalline samples in 10 min enabled one to distinguish alpha- and beta-forms of Cu(II)(8-quinolinol)2 on the basis of shift positions and line widths.     

Direct NMR resonance assignments of the active site histidine residue in Escherichia coli thioesterase I/protease I/lysophospholipase L1

Owing to the hydrogen-bond interaction and rapid exchange rate with the bulk water, the transverse relaxation time for the N(delta1)-H proton of the catalytic histidine in Escherichia coli thioesterase I/protease I/lysophospholipase L1 (TEP-I) is rather short. Because of its catalytic importance, it is desirable to detect and assign this proton resonance. In this paper, we report the first direct NMR correlation between the short-lived N(delta1)-H proton and its covalently attached N(delta1)-nitrogen of the catalytic His157 residue in E. coli thioesterase/protease I. We have used gradient-enhanced jump-return spin-echo HMQC (GE-JR SE HMQC) to obtain a direct correlation between the short-lived N(delta1)-H proton and its covalently attached N(delta1)-nitrogen. The sensitivity of detection for the short-lived N(delta1)-H proton was enhanced substantially by improved water suppression, in particular, the suppression of radiation damping via pulsed field gradients.     

Magic angle spinning NMR experiments for structural studies of differentially enriched protein interfaces and protein assemblies

Protein-protein interactions play vital roles in numerous biological processes. These interactions often result in formation of insoluble and noncrystalline protein assemblies. Solid-state NMR spectroscopy is rapidly emerging as a premier method for structural analysis of such systems. We introduce a family of two-dimensional magic angle spinning (MAS) NMR experiments for structural studies of differentially isotopically enriched protein assemblies. Using 1-73((13)C,(15)N)/74-108((15)N) labeled thioredoxin reassembly, we demonstrate that dipolar dephasing followed by proton-assisted heteronuclear magnetization transfer yields long-range (15)N-(13)C correlations arising exclusively from the interfaces formed by the pair of differentially enriched complementary fragments of thioredoxin. Incorporation of dipolar dephasing into the (15)N proton-driven spin diffusion and into the (1)H-(15)N FSLG-HETCOR sequences permits (1)H and (15)N resonance assignments of the 74-108((15)N) enriched C-terminal fragment of thioredoxin alone. The differential isotopic labeling scheme and the NMR experiments demonstrated here allow for structural analysis of both the interface and each interacting protein. Isotope editing of the magnetization transfers results in spectral simplification, and therefore larger protein assemblies are expected to be amenable to these experiments.     

Excitation of magnetization using a modulated radiation damping field

In this work, pulsed-field gradients are used to modulate the radiation damping field generated by the detection coil in an NMR experiment in order that spins with significantly different chemical shifts can affect one another via the radiation damping field. Experiments performed on solutions of acetone/water and acetone/DMSO/water demonstrate that spins with chemical shift differences much greater than the effective radiation damping field strength can still be coupled by modulating the radiation damping field. Implications for applications in high-field NMR and for developing sensitive magnetization detectors are discussed.     

Molecular organization of hydrophobic molecules and co-adsorbed water in SBA-15 ordered mesoporous silica material

The purpose of this study was to improve our understanding of the molecular organization of hydrophobic guest molecules in the presence of co-adsorbed water inside SBA-15 ordered mesoporous silica material. Understanding this adsorption competition is essential in the development of applications of controlled adsorption and desorption. The poorly water soluble drug compound itraconazole and the fluorescent probe Nile red were selected for the study. The interaction between itraconazole and SBA-15 was investigated using FT-IR, (1)H MAS NMR and (29)Si MAS NMR spectroscopy, by determination of adsorption isotherms and release kinetics in simulated gastric fluid. The distribution and migration of the hydrophobic fluorescent probe Nile red was visualized in situ using confocal fluorescence microscopy. For both molecules, there was a pronounced influence of the co-adsorbed water on adsorption, hydrophobic aggregation and migration in SBA-15 pores. These insights contribute to the development of practical methods for loading ordered mesoporous silica materials with hydrophobic molecules.     

Sensitivity improvement of 1H-15N cross-polarization at high MAS frequency applied to NMR structural characterization of organic solids

(15)N CP/MAS solid state NMR should be a method of choice to obtain essential structural information on organic materials containing nitrogen atoms. However, the technique is generally not selected for the characterization of non-labelled chemical compounds, which represents the most common situation encountered by chemists. Actually, due to the poor sensitivity of (15)N the method is time-consuming and a very fine calibration is often a prerequisite to reach a sufficient signal/noise. The main drawback comes from the weakness of (15)N-(1)H dipolar couplings which leads to a splitting of the static Hartman Hahn condition into very narrow sideband conditions under MAS. Practically, it is more difficult to obtain a high enough CP transfer level on (15)N for the entire spectrum than on other more conventional nuclei like (13)C. An experimental investigation of the CP efficiency using the ramp and adiabatic CP transfer experiments is here proposed. Preliminary adjustments of experimental settings were first made on an (15)N-labeled substituted heterocyclic model system, and then applied to several other organic compounds. Particular attention was paid to the detection of non-protonated nitrogen atoms with a significant chemical shift anisotropy, which represented the least favourable case. It was experimentally demonstrated that, for these atoms, the adiabatic passage provided a much higher transfer level than the more conventional ramp sequence leading to an enhancement factor of up to 3.5 at a MAS frequency of 30 kHz. The resulting sensitivity rendered possible the detection of non-protonated nitrogen atoms at natural abundance with 2.5-mm rotors at 9.4 T.     

Magic-angle-spinning-induced local ordering in polymer electrolytes and its effects on solid-state diffusion and relaxation NMR measurements

Magic-angle-spinning (MAS) enhances sensitivity and resolution in solid-state nuclear magnetic resonance (NMR) measurements. MAS is obtained by aerodynamic levitation and drive of a rotor, which results in large centrifugal forces that may affect the physical state of soft materials, such as polymers, and subsequent solid-state NMR measurements. Here, we investigate the effects of MAS on the solid-state NMR measurements of a polymer electrolyte for lithium-ion battery applications, poly(ethylene oxide) (PEO) doped with the lithium salt LiTFSI. We show that MAS induces local chain ordering, which manifests itself as characteristic lineshapes with doublet-like splittings in subsequent solid-state ¹ H, ⁷ Li, and ¹⁹ F static NMR spectra characterizing the PEO chains and solvated ions. MAS results in distributions of stresses and hence local chain orientations within the rotor, yielding distributions in the local magnetic susceptibility tensor that give rise to the observed NMR anisotropy and lineshapes. The effects of MAS were investigated on solid-state ⁷ Li and ¹⁹ F pulsed-field-gradient (PFG) diffusion and ⁷Li longitudinal relaxation NMR measurements. Activation energies for ion diffusion were affected modestly by MAS. ⁷Li longitudinal relaxation rates, which are sensitive to lithium-ion dynamics in the nanosecond regime, were essentially unchanged by MAS. We recommend that NMR researchers studying soft polymeric materials use only the spin rates necessary to achieve the desired enhancements in sensitivity and resolution, as well as acquire static NMR spectra after MAS experiments to reveal any signs of stress-induced local ordering.     

Heating and temperature gradients of lipid bilayer samples induced by RF irradiation in MAS solid‐state NMR experiments

The MAS solid‐state NMR has been a powerful technique for studying membrane proteins within the native‐like lipid bilayer environment. In general, RF irradiation in MAS NMR experiments can heat and potentially destroy expensive membrane protein samples. However, under practical MAS NMR experimental conditions, detailed characterization of RF heating effect of lipid bilayer samples is still lacking. Herein, using ¹H chemical shift of water for temperature calibration, we systematically study the dependence of RF heating on hydration levels and salt concentrations of three lipids in MAS NMR experiments. Under practical ¹H decoupling conditions used in biological MAS NMR experiments, three lipids show different dependence of RF heating on hydration levels as well as salt concentrations, which are closely associated with the properties of lipids. The maximum temperature elevation of about 10 °C is similar for the three lipids containing 200% hydration, which is much lower than that in static solid‐state NMR experiments. The RF heating due to salt is observed to be less than that due to hydration, with a maximum temperature elevation of less than 4 °C in the hydrated samples containing 120 mmol l^?1 of salt. Upon RF irradiation, the temperature gradient across the sample is observed to be greatly increased up to 20 °C, as demonstrated by the remarkable broadening of ¹H signal of water. Based on detailed characterization of RF heating effect, we demonstrate that RF heating and temperature gradient can be significantly reduced by decreasing the hydration levels of lipid bilayer samples from 200% to 30%. Copyright © 2016 John Wiley & Sons, Ltd.     

Rapid sample-mixing technique for transient NMR and photo-CIDNP spectroscopy: applications to real-time protein folding

We describe the development and application of a novel rapid sample-mixing technique for real-time NMR (nuclear magnetic resonance) spectroscopy. The apparatus consists of an insert inside a conventional NMR tube coupled to a rapid injection syringe outside the NMR magnet. Efficient and homogeneous mixing of solutions in the NMR tube is achieved with a dead time of tens of milliseconds, without modification of the NMR probe or additional hardware inside the magnet. Provision is made for the inclusion of an optical fiber to allow in situ laser irradiation of samples, for example to generate photo-CIDNP (chemically induced dynamic nuclear polarization). An NMR water suppression method has been implemented to allow experiments in H(2)O as well as in deuterated solvents. The performance of the device has been tested and optimized by a variety of methods, including sensitive detection of residual pH gradients and the use of NMR imaging to monitor the extent of mixing in real time. The potential utility of this device, in conjunction with the sensitivity and selectivity of photo-CIDNP, is demonstrated by experiments on the protein hen lysozyme. These measurements involve the direct detection of spectra during real-time refolding, and the use of CIDNP pulse labeling to study a partially unfolded state of the protein under equilibrium conditions. Magnetization transfer from this disordered state to the well-characterized native state provides evidence for the remarkable persistence of nativelike elements of structure under conditions in which the protein is partially denatured and aggregation prone.     

Multinuclear NMR investigations of the oxygen, water, and hydroxyl environments in sodium hexaniobate

Solid-state (1)H, (17)O MAS NMR, (1)H-(93)Nb TRAPDOR NMR, and (1)H double quantum 2D MAS NMR experiments were used to characterize the oxygen, water, and hydroxyl environments in the monoprotonated hexaniobate material, Na(7)[HNb(6)O(19)].15H(2)O. These solid-state NMR experiments demonstrate that the proton is located on the bridging oxygen of the [Nb(6)O(19)](8-) cluster. The solid-state NMR results also show that the NbOH protons are spatially isolated from similar protons, but undergo proton exchange with the water species located in the crystal lattice. On the basis of double quantum (1)H MAS NMR measurements, it was determined that the water species in the crystal lattice have restricted motional dynamics. Two-dimensional (1)H-(17)O MAS NMR correlation experiments show that these restricted waters are preferentially associated with the bridging oxygen. Solution (17)O NMR experiments show that the hydroxyl proton is also attached to the bridging oxygen for the compound in solution. In addition, solution (17)O NMR kinetic studies for the hexaniobate allowed the measurement of relative oxygen exchange rates between the bridging, terminal, and hydroxyl oxygen and the oxygen of the solvent as a function of pH and temperature. These NMR experiments are some of the first investigations into the proton location, oxygen and proton exchange processes, and water dynamics for a base stable polyoxoniobate material, and they provide insight into the chemistry and reactivity of these materials.     

Natural-abundance solid-state 2H NMR spectroscopy at high magnetic field

High-resolution solid-state (2)H NMR spectroscopy provides a method for measuring (1)H NMR chemical shifts in solids and is advantageous over the direct measurement of high-resolution solid-state (1)H NMR spectra, as it requires only the application of routine magic angle sample spinning (MAS) and routine (1)H decoupling methods, in contrast to the requirement for complex pulse sequences for homonuclear (1)H decoupling and ultrafast MAS in the case of high-resolution solid-state (1)H NMR. However, a significant obstacle to the routine application of high-resolution solid-state (2)H NMR is the very low natural abundance of (2)H, with the consequent problem of inherently low sensitivity. Here, we explore the feasibility of measuring (2)H MAS NMR spectra of various solids with natural isotopic abundances at high magnetic field (850 MHz), focusing on samples of amino acids, peptides, collagen, and various organic solids. The results show that high-resolution solid-state (2)H NMR can be used successfully to measure isotropic (1)H chemical shifts in favorable cases, particularly for mobile functional groups, such as methyl and -N(+)H(3) groups, and in some cases phenyl groups. Furthermore, we demonstrate that routine (2)H MAS NMR measurements can be exploited for assessing the relative dynamics of different functional groups in a molecule and for assessing whole-molecule motions in the solid state. The magnitude and field-dependence of second-order shifts due to the (2)H quadrupole interaction are also investigated, on the basis of analysis of simulated and experimental (1)H and (2)H MAS NMR spectra of fully deuterated and selectively deuterated samples of the α polymorph of glycine at two different magnetic field strengths.     

Membrane-bound conformation of peptaibols with methyl-deuterated alpha-amino isobutyric acids by 2H magic angle spinning solid-state NMR spectroscopy

We present the use of 2H magic-angle spinning (MAS) NMR on methyl-deuterated alpha-amino isobutyric acid (Aib) as a new method to obtain fast and accurate structural constraints on peptaibols in membrane-bound environments. Using nonoriented vesicle-reconstituted samples we avoid the delicate preparation of oriented samples, and the use of MAS ensures high sensitivity and thereby very fast acquisition of experimental spectra. Furthermore, given the high content ( approximately 40%) of Aib in peptaibols and the fact that the amino acid Aib may be synthesized from cheap starting materials, even in the case of 2H, 13C, or 15N labeling, this method is ideally suited for studies of the membrane-bound conformation of peptaibols.     

Charakterisierung der Protonen in polykristallinen Parawolframaten durch 1H‐MAS‐NMR‐Untersuchungen

Characterization of the Protons in Polycrystalline Paratungstates using ¹H MAS NMR Investigations ¹H MAS NMR experiments are used to characterize the non‐acid protons of the anions in polycrystalline paratungstates by means of the measured isotropic chemical shift values. The investigation of various hydrates of ammonium paratungstate allows a direct proof of protons in NH₄ ions and in water molecules while protons of the anions are not detectable. However, for both the potassium and the sodium paratungstates ¹H MAS NMR investigations detected the protons of water molecules and the non‐acid protons of the paratungstate anions. Additional ¹H broad‐line NMR experiments at 173 K support the interpretation of the results obtained by the ¹H MAS NMR investigations. For the NMR signal of the non‐acid protons of the paratungstate anion in the ¹H MAS NMR spectra of the potassium salt line‐splitting appears. This refers to the existence of two nonidentical positions of the protons in the crystal lattice and is in agreement with the results of the X‐ray structural analysis.     

Application of automated eightfold suppression of water and ethanol signals in 1H NMR to provide sensitivity for analyzing alcoholic beverages

The 400 MHz (1)H NMR analysis of alcoholic beverages using standard pulse programs lacks the necessary sensitivity to detect minor constituents such as methanol, acetaldehyde or ethyl acetate. This study investigates the application of a shaped pulse sequence during the relaxation delay to suppress the eight (1)H NMR frequencies of water and ethanol (the OH singlet of both water and ethanol, as well as the CH(2) quartet and CH(3) triplet of ethanol). The sequence of reference measurement for frequency determination followed by the suppression experiment is controlled by a macro in the acquisition software so that a measurement under full automation is possible (12 min per sample total time). Additionally, sample preparation was optimized to avoid precipitation, which is facilitated by 1:1 dilution with ethanol and pH 7.4 buffer. Compared with the standard water presaturation pulse program, the eightfold suppression allowed a significantly higher setting of receiver gain without receiver overflow, which significantly increased the signal-to-noise ratio by an average factor of 10. The signal intensities increased by a factor of 20. The resulting limits of detection (below 1 g/hl of pure alcohol) now allow the control of legal requirements for minor compounds in alcoholic beverages.