Observed NMR Signals Research Articles

Multi‐Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In‐Cell NMR

AbstractGoverning function, half‐life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in‐cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co‐existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

Multi-Dimensional Structure and Dynamics Landscape of Proteins in Mammalian Cells Revealed by In-Cell NMR.

Governing function, half-life and subcellular localization, the 3D structure and dynamics of proteins are in nature constantly changing in a tightly regulated manner to fulfill the physiological and adaptive requirements of the cells. To find evidence for this hypothesis, we applied in-cell NMR to three folded model proteins and propose that the splitting of cross peaks constitutes an atomic fingerprint of distinct structural states that arise from multiple target binding co-existing inside mammalian cells. These structural states change upon protein loss of function or subcellular localisation into distinct cell compartments. In addition to peak splitting, we observed NMR signal intensity attenuations indicative of transient interactions with other molecules and dynamics on the microsecond to millisecond time scale.

Hyperpolarized Solution-State NMR Spectroscopy with Optically Polarized Crystals.

Nuclear spin hyperpolarization provides a promising route to overcome the challenges imposed by the limited sensitivity of nuclear magnetic resonance. Here we demonstrate that dissolution of spin-polarized pentacene-doped naphthalene crystals enables transfer of polarization to target molecules via intermolecular cross-relaxation at room temperature and moderate magnetic fields (1.45 T). This makes it possible to exploit the high spin polarization of optically polarized crystals, while mitigating the challenges of its transfer to external nuclei. With this method, we inject the highly polarized mixture into a benchtop NMR spectrometer and observe the polarization dynamics for target 1H nuclei. Although the spectra are radiation damped due to the high naphthalene magnetization, we describe a procedure to process the data to obtain more conventional NMR spectra and extract the target nuclei polarization. With the entire process occurring on a time scale of 1 min, we observe NMR signals enhanced by factors between -200 and -1730 at 1.45 T for a range of small molecules.

Observability of Paramagnetic NMR Signals at over 10 000 ppm Chemical Shifts

AbstractWe report an experimental observation of 31P NMR resonances shifted by over 10 000 ppm (meaning percent range, and a new record for solutions), and similar 1H chemical shifts, in an intermediate‐spin square planar ferrous complex [tBu(PNP)Fe‐H], where PNP is a carbazole‐based pincer ligand. Using a combination of electronic structure theory, nuclear magnetic resonance, magnetometry, and terahertz electron paramagnetic resonance, the influence of magnetic anisotropy and zero‐field splitting on the paramagnetic shift and relaxation enhancement is investigated. Detailed spin dynamics simulations indicate that, even with relatively slow electron spin relaxation (T1 ≈10−11 s), it remains possible to observe NMR signals of directly metal‐bonded atoms because pronounced rhombicity in the electron zero‐field splitting reduces nuclear paramagnetic relaxation enhancement.

Observability of Paramagnetic NMR Signals at over 10 000 ppm Chemical Shifts.

We report an experimental observation of 31P NMR resonances shifted by over 10 000 ppm (meaning percent range, and a new record for solutions), and similar 1H chemical shifts, in an intermediate‐spin square planar ferrous complex [ tBu(PNP)Fe‐H], where PNP is a carbazole‐based pincer ligand. Using a combination of electronic structure theory, nuclear magnetic resonance, magnetometry, and terahertz electron paramagnetic resonance, the influence of magnetic anisotropy and zero‐field splitting on the paramagnetic shift and relaxation enhancement is investigated. Detailed spin dynamics simulations indicate that, even with relatively slow electron spin relaxation (T 1 ≈10−11 s), it remains possible to observe NMR signals of directly metal‐bonded atoms because pronounced rhombicity in the electron zero‐field splitting reduces nuclear paramagnetic relaxation enhancement.

Al/Ga-Doped Li7La3Zr2O12 Garnets as Li-Ion Solid-State Battery Electrolytes: Atomistic Insights into Local Coordination Environments and Their Influence on 17O, 27Al, and 71Ga NMR Spectra.

Li7La3Zr2O12 (LLZO) garnets are among the most promising solid electrolytes for next-generation all-solid-state Li-ion battery applications due to their high stabilities and ionic conductivities. To help determine the influence of different supervalent dopants on the crystal structure and site preferences, we combine solid-state 17O, 27Al, and 71Ga magic angle spinning (MAS) NMR spectroscopy and density-functional theory (DFT) calculations. DFT-based defect configuration analysis for the undoped and Al and/or Ga-doped LLZO variants uncovers an interplay between the local network of atoms and the observed NMR signals. Specifically, the two characteristic features observed in both 27Al and 71Ga NMR spectra result from both the deviations in the polyhedral coordination/site-symmetry within the 4-fold coordinated Li1/24d sites (rather than the doping of the other Li2/96h or La sites) and with the number of occupied adjacent Li2 sites that share oxygen atoms with these dopant sites. The sharp 27Al and 71Ga resonances arise from dopants located at a highly symmetric tetrahedral 24d site with four corner-sharing LiO4 neighbors, whereas the broader features originate from highly distorted dopant sites with fewer or no immediate LiO4 neighbors. A correlation between the size of the 27Al/71Ga quadrupolar coupling and the distortion of the doping sites (viz. XO4/XO5/XO6 with X = {Al/Ga}) is established. 17O MAS NMR spectra for these systems provide insights into the oxygen connectivity network: 17O signals originating from the dopant-coordinating oxygens are resolved and used for further characterization of the microenvironments at the dopant and other sites.

Protein Structure Determination in Living Cells.

To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes but not ‘direct’ data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works.

Structural Analysis of the Terminal Groups in Commercial Hevea Natural Rubber by 2D-NMR with DOSY Filters and Multiple-WET Methods Using Ultrahigh-Field NMR.

The terminal groups of natural rubber (NR) are widely believed to play a crucial role in defining the excellent mechanical and other physical properties of processed NR products. Despite their presumed importance, the chemical structures of the terminal groups are elusive in widely used NR species with a high degree of polymerization, such as Hevea natural rubber (H-NR). In previous studies, structural analysis by solution NMR has been carried out on the terminal units of NR after chemical treatment involving chemical alterations, such as deproteinization with enzymes and other chemicals. However, there is concern that such chemical treatments may alter the properties of the terminal units. In this study, we established an NMR-based approach to analyze the structures of the terminal units in commercial H-NR without any chemical treatments, or with only a mild treatment of some samples, such as acetone extraction for removing the impurities. To suppress the signals of low-molecular-weight impurities, we have developed methods combining DOSY-based diffusion filters with multiple-WET (MWET) 2D-NMR, which we introduced previously to suppress strong signals from main-chain of polymer and solvents (Tanaka et al. Macromolecules, 2016, 49, 5750-5754). Using the new method and MWET 2D-NMR methods with high-field NMR at a 1H frequency of 900 MHz, we observed NMR signals of the terminal units of chemically untreated commercial H-NR for the first time. The NMR results for eight commercial H-NR samples consistently demonstrated the presence of at least five kinds of terminating-end (α-terminus) units of the H-NR polymer chain in addition to NMR signals for the initiating-end (ω-terminus) units. Our NMR analyses revealed for the first time that none of the α-terminal groups form a phosphate ester.

In vivo Observation of Tree Drought Response with Low-Field NMR and Neutron Imaging.

Using a simple low-field NMR system, we monitored water content in a living tree in a greenhouse over 2 months. By continuously running the system, we observed changes in tree water content on a scale of half an hour. The data showed a diurnal change in water content consistent both with previous NMR and biological observations. Neutron imaging experiments show that our NMR signal is primarily due to water being rapidly transported through the plant, and not to other sources of hydrogen, such as water in cytoplasm, or water in cell walls. After accounting for the role of temperature in the observed NMR signal, we demonstrate a change in the diurnal signal behavior due to simulated drought conditions for the tree. These results illustrate the utility of our system to perform noninvasive measurements of tree water content outside of a temperature controlled environment.

Toward recording 33S NMR spectra of cross‐linked elastomers: Relaxation dynamics perspective

AbstractWe discuss from the perspective of relaxation dynamics the feasibility of recording NMR spectra of 33S nuclei bound to cross‐link junctions in cross‐linked elastomers (i.e. vulcanized rubber). Assuming the quadrupolar relaxation mechanism due to fluctuating electric field gradients, the calculations of transverse and longitudinal 33S relaxation times encompassing a large range of correlation times, static magnetic fields, and quadrupolar coupling constants reveal that both relaxation processes will constitute significant bottlenecks in recording the 33S spectra of cross‐linked elastomers inherently endowed with a large distribution of motional modes. In the rubbery state, the observable 33S NMR signal will result from motionally averaged, central transition magnetization of the 33S nuclei in cross‐link junctions involved in large amplitude, high‐frequency motions.

Long live the spin

A technique for protecting out-of-equilibrium nuclear spin states from thermalization while offering a route to converting them into observable NMR signal is an important contribution to a field that welcomes every bit of extra signal.

Observable NMR signal from circulating current order in YBCO

Assuming, as suggested by recent neutron scattering experiments, that a broken symmetry state with orbital current order occurs in the pseudo-gap phase of the cuprate superconductors, we show that there must be associated equilibrium magnetic fields at various atomic sites in the unit cell, which should be detectable by NMR experiments.

NMR quantitation: influence of RF inhomogeneity.

The NMR peak integral is ideally linearly dependent on the sine of excitation angle (θ), which has provided unsurpassed flexibility in quantitative NMR by allowing the use of a signal of any concentration as the internal concentration reference. Controlling the excitation angle is particularly critical for solvent proton concentration referencing to minimize the negative impact of radiation damping, and to reduce the risk of receiver gain compression. In practice, due to the influence of RF inhomogeneity for any given probe, the observed peak integral is not exactly proportional to sin θ. To evaluate the impact quantitatively, we introduce a RF inhomogeneity factor I(θ) as a function of the nominal pulse excitation angle and propose a simple calibration procedure. Alternatively, I(θ) can be calculated from the probe's RF profile, which can be readily obtained as a gradient image of an aqueous sample. Our results show that without consideration of I(θ), even for a probe with good RF homogeneity, up to 5% error can be introduced due to different excitation pulse angles used for the analyte and the reference. Hence, a simple calibration of I(θ) can eliminate such errors and allow an accurate description of the observed NMR signal's dependence on the excitation angle in quantitative analysis.

DFT study of possible lattice defects in methane-hydrate and their appearance in 13C NMR spectra

The effect of the possible cell defects in the methane-hydrate crystal on the chemical shift of the carbon core has been investigated by DFT calculations. The clathrate structure was modeled as a water monolayer, in mono and bicavital clusters. The results show that the radius of the clathrate cell correlates with the chemical shielding of the carbon core. The calculated chemical shifts for the most stable clusters are in good agreement with the observed NMR signals supporting the previous spectral assignment. The occupancy of a neighbouring cell in a bicavital cluster has a small effect on the calculated NMR shift.

Receiver gain function: the actual NMR receiver gain

The observed NMR signal size depends on the receiver gain parameter. We propose a receiver gain function to characterize how much the raw FID is amplified by the receiver as a function of the receiver gain setting. Although the receiver is linear for a fixed gain setting, the actual gain of the receiver may differ from what the gain setting suggests. Nevertheless, for a given receiver, we demonstrate that the receiver gain function can be calibrated. Such a calibration enables accurate comparison of separately acquired NMR signals in quantitative analysis, which frequently requires different receiver gain settings to avoid receiver saturation or achieve optimum sensitivity. The application of receiver gain function, along with the definition of receiving efficiency, allows easy concentration determination by a single internal or external concentration reference.

NMR spectral enantioresolution of spirobrassinin and 1-methoxyspirobrassinin enantiomers using ( S)-(−)-ethyl lactate and modeling of spirobrassinin self-association for rationalization of its self-induced diastereomeric anisochromism (SIDA) and enantiomer self-disproportionation on achiral-phase chromatography (ESDAC) phenomena

The phytoalexin spirobrassinin is known to exhibit the unusual phenomenon of enantiomer self-disproportionation on achiral-phase chromatography (ESDAC) whereby the enantiomeric composition of a non-racemic mixture varies across the eluting chromatographic peak. However, spirobrassinin is exceptional since both early and late fractions are enantioenriched in comparison to the starting enantiomeric composition. This behavior is rationalized by the formation of two different complexes, substantiated by DFT calculations at the B3LYP/TZVP level of theory, both of which are in dynamic equilibrium with the free molecules. Concomitant with complexation under chromatographic conditions, complexation was also evident by NMR including the observation of distinct NMR signals for each enantiomer for some spins in a non-racemic mixture—the phenomenon of self-induced diastereomeric anisochromism (SIDA). Enantioresolution of various 1H, 13C and 15N NMR signals were also induced for the enantiomers of spirobrassinin and another phytoalexin, 1-methoxyspirobrassinin, by application of the chiral solvating agent ( S)-(−)-ethyl lactate, a cheap reagent with potentially broad applications. For spirobrassinin, the observed NMR signals were evaluated against values calculated using the GIAO method at the B3LYP/cc-pVTZ level of theory.

Dynamic Nuclear Polarization with a Rigid Biradical

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether.

Dynamic Nuclear Polarization with a Rigid Biradical

AbstractEine starre Spirobrücke verknüpft zwei 2,2,6,6‐Tetramethylpiperidin‐1‐oxyl(TEMPO)‐Einheiten in einem neuen diradikalischen Polarisierungsmittel für DNP‐Experimente. Die beobachteten NMR‐Signalintensitäten sind 1.4mal größer als für TOTAPOL, ein zuvor beschriebenes Diradikal aus zwei TEMPO‐Einheiten und einer flexiblen Brücke.magnified image

The noncatalytic triad of α‐amylases: A novel structural motif involved in conformational stability

Chloride-activated alpha-amylases contain a noncatalytic triad, independent of the glycosidic active site, perfectly mimicking the catalytic triad of serine-proteases and of other active serine hydrolytic enzymes. Mutagenesis of Glu, His, and Ser residues in various alpha-amylases shows that this pattern is a structural determinant of the enzyme conformation that cannot be altered without losing the intrinsic stability of the protein. (1)H-(15)N NMR spectra of a bacterial alpha-amylase reveal proton signals that are identical with the NMR signature of catalytic triads and especially a deshielded proton involving a protonated histidine and displaying properties similar to that of a low barrier hydrogen bond. It is proposed that the H-bond between His and Glu of the noncatalytic triad is an unusually strong interaction, responsible for the observed NMR signal and for the weak stability of the triad mutants. Furthermore, a stringent template-based search of the Protein Data Bank demonstrated that this motif is not restricted to alpha-amylases, but is also found in 80 structures from 33 different proteins, amongst which SH2 domain-containing proteins are the best representatives.

Toward direct neural current imaging by resonant mechanisms at ultra-low field

A variety of techniques have been developed to noninvasively image human brain function that are central to research and clinical applications endeavoring to understand how the brain works and to detect pathology (e.g. epilepsy, schizophrenia, etc.). Current methods can be broadly divided into those that rely on hemodynamic responses as indicators of neural activity (e.g. fMRI, optical, and PET) and methods that measure neural activity directly (e.g. MEG and EEG). The approaches all suffer from poor temporal resolution, poor spatial localization, or indirectly measuring neural activity. It has been suggested that the proton spin population will be altered by neural activity resulting in a measurable effect on the NMR signal that can be imaged by MRI methods. We present here the physical basis and experimental evidence for the resonant interaction between magnetic fields such as those arising from neural activity, with the spin population in ultra-low field (μT) NMR experiments. We demonstrate through the use of current phantoms that, in the case of correlated zero-mean current distributions such as those one might expect to result from neural activity, resonant interactions will produce larger changes in the observed NMR signal than dephasing. The observed resonant interactions reported here might one day form the foundation of a new functional neuroimaging modality ultimately capable of simultaneous direct neural activity and brain anatomy tomography.