NMR Sample Research Articles

How (and why) to determine NMR spectrometer’s noise figure?

Abstract In the last decade or two, it seems that the trend of technological advance in NMR spectroscopy cannot follow the trend of desire to measure NMR samples with gradually lower response signals. Recently, an accurate noise model, based on the concept of noise figure, of the most sensitive part of the NMR spectroscopy system from the aspect of noise, which is its probe-to-spectrometer receiving chain, was introduced. The main purpose of this model is to optimize the used NMR spectroscopy system and, ultimately, enable measuring NMR samples with even lower response signals than the ones measured today. All the parameters of the NMR spectroscopy system, used in the introduced model, can be easily measured using vector network analyzers and noise figure meters, or can be found in the datasheets of the respective elements, except the spectrometer’s receiving chain noise figure. In this contribution, the process of spectrometer’s receiving chain noise figure measurement, performed using the Twice Power Method, is described. A block diagram representation of the spectrometer’s receiving chain is presented here, as well as its approximative model. The respective noise figure measurement results, which are also presented, explain the general tendency of using the mid-range of the spectrometer’s gain control level when performing the actual NMR measurements.

Flavodoxin in a binary surfactant system consisting of the nonionic 1-decanoyl-rac-glycerol and the zwitterionic lauryldimethylamine-N-oxide: Molecular dynamics simulation approach

Due to the short time constant of the spin-spin relaxation process, there is a limitation in the preparation of NMR sample solution for large proteins. To overcome this problem, reverse micelle systems are used. Here, molecular dynamics simulation was used to study the structure of flavodoxin in a quaternary mixture of 1-decanoyl-rac-glycerol, lauryldimethylamine-N-oxide, pentane and hexanol. Hexanol was used as co-solvent. Simulations were performed at three different co-solvent concentrations. The proportion of components in the mixture was selected according to experimental conditions. For comparison, simulation of flavodoxin in water was also performed. The simulation results show that the C$\alpha$-RMSD for the protein in water is less than for the surfactant mixture. Also, the radius of gyration of flavodoxin increased in the presence of surfactants. The distance between the two residues trp-57 and phe-94, as a measure of protein activity, was obtained from the simulations. The results showed that in the surfactant mixtures this distance increases. Analysis of the secondary structure of the protein shows that the N-terminal part of the flavodoxin is more affected by surfactants. The flavodoxin diffusion coefficient in the surfactant mixture decreased in relation to its diffusion coefficient in water.

Ferromagnetic contamination of ultra-low-field-NMR sample containers. Quantification of the problem and possible solutions

The presence of a weak remanence in Ultra-Low-Field (ULF) NMR sample containers is investigated on the basis of proton precession. The high-sensitivity magnetometer used for the NMR detection, enables simultaneously the measurement of the static field produced in the sample proximity by ferromagnetic contaminants. The presence of the latter is studied by high resolution chemical analyses of the surface, based on X-ray fluorescence spectroscopy and secondary ions mass spectroscopy. Methodologies to reduce the contamination are explored and characterized. This study is of relevance in any ULF-NMR experiment, as in the ULF regime spurious ferromagnetism becomes easily a dominant cause of artefacts.

Phosphorus NMR and Its Application to Metabolomics.

Stable isotopes are routinely employed by NMR metabolomics to highlight specific metabolic processes and to monitor pathway flux. 13C-carbon and 15N-nitrogen labeled nutrients are convenient sources of isotope tracers and are commonly added as supplements to a variety of biological systems ranging from cell cultures to animal models. Unlike 13C and 15N, 31P-phosphorus is a naturally abundant and NMR active isotope that does not require an external supplemental source. To date, 31P NMR has seen limited usage in metabolomics because of a lack of reference spectra, difficulties in sample preparation, and an absence of two-dimensional (2D) NMR experiments, but 31P NMR has the potential of expanding the coverage of the metabolome by detecting phosphorus-containing metabolites. Phosphorylated metabolites regulate key cellular processes, serve as a surrogate for intracellular pH conditions, and provide a measure of a cell's metabolic energy and redox state, among other processes. Thus, incorporating 31P NMR into a metabolomics investigation will enable the detection of these key cellular processes. To facilitate the application of 31P NMR in metabolomics, we present a unified protocol that allows for the simultaneous and efficient detection of 1H-, 13C-, 15N-, and 31P-labeled metabolites. The protocol includes the application of a 2D 1H-31P HSQC-TOCSY experiment to detect 31P-labeled metabolites from heterogeneous biological mixtures, methods for sample preparation to detect 1H-, 13C-, 15N-, and 31P-labeled metabolites from a single NMR sample, and a data set of one-dimensional (1D) 31P NMR and 2D 1H-31P HSQC-TOCSY spectra of 38 common phosphorus-containing metabolites to assist in metabolite assignments.

Optimizing the α1B-adrenergic receptor for solution NMR studies

Sample preparation for NMR studies of G protein-coupled receptors faces special requirements: Proteins need to be stable for prolonged measurements at elevated temperatures, they should ideally be uniformly labeled with the stable isotopes 13C, 15N, and all carbon-bound protons should be replaced by deuterons. In addition, certain NMR experiments require protonated methyl groups in the presence of a perdeuterated background. All these requirements are most easily satisfied when using Escherichia coli as the expression host. Here we describe a workflow, starting from a temperature-stabilized mutant of the α1B-adrenergic receptor, obtained using the CHESS methodology, into an even more stable species, in which flexible parts from termini were removed and the intracellular loop 3 (ICL3) was stabilized against proteolytic cleavage. The yield after purification corresponds to 1–2 mg/L of D2O culture. The final purification step is ligand-affinity chromatography to ensure that only well-folded ligand-binding protein is isolated. Proper selection of detergent has a remarkable influence on the quality of NMR spectra. All optimization steps of sequence and detergent are monitored on a small scale by monitoring the melting temperature and long-term thermal stability to allow for screening of many conditions. The stabilized mutant of the α1B-adrenergic receptor was additionally incorporated in nanodiscs, but displayed slightly inferior spectra compared to a sample in detergent micelles. Finally, both [15N,1H]- as well as [13C,1H]-HSQC spectra are shown highlighting the high quality of the final NMR sample. Importantly, the quality of [13C,1H]-HSQC spectra indicates that the so prepared receptor could be used for studying side-chain dynamics.

Flanking Regions Determine the Structure of the Poly-Glutamine in Huntingtin through Mechanisms Common among Glutamine-Rich Human Proteins

The causative agent of Huntington's disease, the poly-Q homo-repeat in the N-terminal region of huntingtin (httex1), is flanked by a 17-residue-long fragment (N17) and a proline-rich region (PRR), which promote and inhibit the aggregation propensity of the protein, respectively, by poorly understood mechanisms. Based on experimental data obtained from site-specifically labeled NMR samples, we derived an ensemble model of httex1 that identified both flanking regions as opposing poly-Q secondary structure promoters. While N17 triggers helicity through a promiscuous hydrogen bond network involving the side chains of the first glutamines in the poly-Q tract, the PRR promotes extended conformations in neighboring glutamines. Furthermore, a bioinformatics analysis of the human proteome showed that these structural traits are present in many human glutamine-rich proteins and that they are more prevalent in proteins with longer poly-Q tracts. Taken together, these observations provide the structural bases to understand previous biophysical and functional data on httex1.

A robust and versatile method for production and purification of large-scale RNA samples for structural biology.

Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.

An allosteric hot spot in the tandem SH2 domain of ZAP‐70 regulates T‐cell signaling

The T‐Cell signaling commences with the binding of Antigen Presenting Cell with T‐cell Receptor (TCR) Complex leading to the recruitment of a non‐receptor tyrosine kinase, Zeta Associated Protein‐70 (ZAP‐70), to the membrane. ZAP‐70 is comprised of the tandem SH2 domain (tSH2), which is made up of N‐terminal SH2 (N‐SH2) and C‐terminal SH2 (C‐SH2) domains, and a carboxy‐terminal kinase domain. The two SH2 domains cooperatively bind to the doubly‐phosphorylated ITAM (ITAM‐Y2P) motif resulting in a conformational rearrangement in the whole of the regulatory module, thereby releasing the autoinhibitory conformation of the kinase domain. However, the structural mechanism of how the two SH2 domain cross‐talk during ITAM‐Y2P binding and dissociation from the membrane are poorly understood. We observed that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly‐phosphorylated ITAM‐ ζ1 (ITAM‐Y2P‐ ζ1) peptide. The C‐SH2 binds first to one of the phosphotyrosine in ITAM‐Y2P motifs, leading to the formation of an encounter complex which further leads to the binding by N‐SH2 domain. NMR titration and Network analysis studies show that an intra‐residue network, which allosterically interconnects the N‐SH2 and C‐SH2 binding sites, plays a central role in ITAM‐Y2P binding. Further, a mutation in the allosteric network (F117A, W165C) uncouples the two binding sites, causing autoimmune arthritis in SKG mice. The allosteric coupling in ZAP‐70 is found to be unique and thus plays a significant role in T‐cell regulation.Support or Funding InformationAuthors are thankful to Prof. John Kuriyan and Prof. David E. Wemmer for access to the 900 MHz NMR spectrometer at the University of California, Berkeley. Dr. Jeffrey G. Pelton and Dr. Patrick R. Visperas at the University of California, Berkeley for NMR data collection and sample preparation. Authors thank Prof. Gautam Basu and Mr. Barun Majumder at Bose Institute, India for access to 700 MHz NMR spectrometer. Authors are thankful to Dr. Ashima Bhattacharjee, Dr. Pradip K. Tarafdar, and Prof. Pradipta Purkayastha for access to ITC and fluorimeter. Authors thank Prof. Giuseppe Melacini and Prof. Maitrayee DasGupta for helpful discussion. The authors thanks research funding from IISER Kolkata, infrastructural facilities supported by IISER Kolkata and DST‐FIST (SR/FST/LS‐II/2017/93(c)). This work is supported by grant from SERB (ECR/2015/000142) and DBT Ramalingaswami Fellowship (BT/RFF/Re‐entry/14/2014) to Rahul Das.Figure 1

Magnetic susceptibility measurement by NMR: 1. The temperature dependence of TMS

Usually a dedicated susceptometer is needed to measure diamagnetism accurately. An NMR spectrometer is more readily available in most chemistry departments but till now has been inaccurate for measuring diamagnetism. An improved NMR method is introduced to measure the magnetic susceptibility, or diamagnetism with similar absolute accuracy as other methods. This is achieved by accurate modelling of the NMR sample shape and response profile of the probe. The new method is validated by comparing the measured diamagnetism of water against the literature standard to within 0.05%. As a first example of its application, the diamagnetism of CDCl3 was measured over a range of temperatures and used to reanalyze earlier measurements of the variation of the chemical shift of tetramethylsilane in CDCl3 against 3He gas. This improved on the accuracy and reliability of the result and will allow, for the first time, accurate studies of the absolute effect of temperature on chemical shift.

Recording In-Cell NMR-Spectra in Living Mammalian Cells.

At the foundation of many cellular processes as well as a large number of diseases is the (mis)folding of important intrinsically disordered proteins (IDPs). Despite tremendous scientific efforts, the factors driving their structural changes within the cellular context remain poorly understood. In-cell NMR spectroscopy enables investigation of IDPs directly in the living eukaryotic cell enabling investigation of its intermolecular interactions and ensuing modifications at an unprecedented atomic resolution. In the following protocol, we describe how to prepare in-cell NMR samples of IDPs within eukaryotic cells and how to measure these in-cell NMR samples of an IDP in its natural environment, the living mammalian cell. Furthermore, we outline a procedure to assess the intracellular recombinant protein concentration of the studied IDP based on in-cell NMR methods. We use α-synuclein as a model protein, but the presented approach is highly modular and therefore should be easily adapted and altered to the desired needs for the studies of different IDPs.

Rapid and Simple Identification and Quantification of Components in Detergent Formulations by Nuclear Magnetic Resonance Spectroscopy

AbstractA rapid, inexpensive, and simple high‐resolution NMR spectroscopic method is outlined for determining the content of surfactants and other low‐molecular‐weight organic compounds in detergent formulations. With simple sample preparation, quantitative results can be obtained from an internal standard and/or the method can be used as a fingerprint analysis of the surfactant composition. The NMR sample is prepared by suspending the detergent sample in deuterated acetic acid and thus dissolving surfactants and other organic compounds. Any content of carbonate will be liberated as CO2, whereas other inorganic materials are removed by centrifugation. From one‐dimensional 1H and two‐dimensional HSQC NMR spectra, the surfactant components and low‐molecular‐weight organic compounds can be identified from reference spectra. Intensities of signature signals in the one‐dimensional 1H NMR spectrum are used for quantification by comparing with an internal standard. Furthermore, it is demonstrated how 31P NMR can be used to identify and quantify phosphonate‐type chelators.

A Triple Layer of Immiscible Solvents for NMR Sample Preparation: Enhanced Sensitivity and Reduced Deuterated Solvent

AbstractNMR spectroscopy is often constrained by lack of sensitivity. A contributing factor towards this reduced sensitivity stems from NMR sample preparation‐ the sample dilution. Organic chemists routinely prepare NMR sample by filling the lowermost 4 cm length of a regular 5 mm 0.d. tube that holds approximately 0.55 ml of a deuterated solvent. This is equivalent to diluting the sample as the signal mainly comes from the central part of length 1.8 cm corresponding to typical coil length of 1.8 cm. The top and bottom part of the sample contributes less signal as they are far from the coil but are crucial for maintaining magnetic field homogeneity. Consequently, the requirement of the expensive deuterated solvent increases. Herein, we explore a way of sample preparation which involves sandwiching a small amount of the deuterated solvent containing the analyte between two non‐deuterated solvents, which are immiscible with the former using a regular 5 mm o.d. tube. The total length is still 4 cm. The analyte now being closer to NMR coil, improves sensitivity and reduces deuterated solvent hitherto required by a factor of two‐fold. 13C and 15N spectra display more than a two‐fold higher signal to noise ratio. 1H, 19F, and 31P spectra also displayed enhanced sensitivity. Examples of various solvent systems designed are detailed here. Two‐dimensional NMR is also provided.

MAS dependent sensitivity of different isotopomers in selectively methyl protonated protein samples in solid state NMR.

Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110kHz and proton content on the spectral quality obtainable on a 1GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.

Partial alignment, residual dipolar couplings and molecular symmetry in solution NMR.

Residual dipolar couplings (RDCs) and residual anisotropic chemical shifts (RACSs) are produced by the partial alignment of solution NMR samples. RDCs and RACSs yield high-resolution structural and dynamic information on the orientation of bonds and chemical groups in molecules. Many molecules form oligomers or have intrinsic symmetries, which may simplify the analysis of their partial alignment datasets. In this report, we explore the theory of partial alignment using an irreducible spherical representation, and we investigate the impact of molecular symmetry on the alignment of molecules. Though previous studies have reported simplified relationships on the partial alignment of molecules bearing different symmetry groups, we show that these simplified relationships may not be universal and only apply to a limited set of systems.

Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.

Nicotine-free, nontransgenic tobacco (Nicotiana tabacum l.) edited by CRISPR-Cas9.

RF shielding and eddy currents in NMR probes.

Shielding of the NMR sample by a thin coating of metal between the sample and the rf coil arises in some geometries. The shielding may be used for rf heating of the sample tube or for a high infrared reflectivity coating in cryoprobe applications. An important application is for a shield that prevents noise from entering the rf probe circuit while allowing pulsed magnetic field gradients or field modulation to pass. We show by simple, approximate derivations that the criterion for shielding is not whether the coating exceeds the classical electromagnetic skin depth δ at the operating frequency (as is often stated), but whether the geometric mean between the thickness and an appropriate radius r exceeds δ. Thus, because r is typically much larger than δ, conducting layers substantially thinner than δ may still be good shields. Measurements are performed at high audio frequencies to confirm the calculations, using geometries relevant to rf saddle coils and to rf solenoids. Measurements of the slowing of the edges of a pulsed field gradient are also in accord with the calculations.

Efficient 263 GHz magic angle spinning DNP at 100 K using solid-state diode sources

The development of new, high-frequency solid-state diode sources capable of operating at 263 GHz, together with an optimized stator design for improved millimeter-wave coupling to the NMR sample, have enabled low-power DNP experiments at 263 GHz/400 MHz. With 250 mW output power, signal enhancements as high as 120 are achieved on standard samples – approximately 1/3 of the maximal enhancement available with high-power gyrotrons under similar conditions. Diode-based sources have a number of advantages over vacuum tube devices: they emit a pure mode, can be rapidly frequency-swept over a wide range of frequencies, have reproducible output power over this range, and have excellent output stability. By virtue of their small size, low thermal footprint, and lack of facility requirements, solid-state diodes are also considerably cheaper to operate and maintain than high-power vacuum tube devices. In light of these features, and anticipating further improvements in terms of available output power, solid-state diodes are likely to find widespread use in DNP and contribute to further advances in the field.

Optimal flip angle for robust practical quantitative NMR measurement using a fixed pulse length.

NMR quantification has been traditionally performed by using internal standards. Although methods using external reference in NMR quantification have been developed, the major obstacles in using external referencing method are the measurement deviations associated with changing sample conditions and the requirement of pulse width calibration for every sample in order to compensate these errors. The calibration process is time consuming and in some cases impossible. We developed a quantitative NMR method fixed pulse length (FIXPUL) for all measurements without sample-by-sample calibration. The method is based on the use of an optimal flip angle calibrated for an external standard so that the quantitative errors associated with the pulse width variations are minimized. FIXPUL can be implemented on most basic NMR spectrometers and is robust and easily automated. The method is applicable to a wide range of solution NMR samples in chemistry, biology, and drug research and discovery.

Composition Determination of Low-Pressure Gas-Phase Mixtures by 1H NMR Spectroscopy.

1H NMR spectroscopy was used to analyze gas-phase mixtures of methane and propane at pressures near 0.1 MPa. The mixtures were prepared gravimetrically and had low uncertainty in their composition. The primary mixture used for this work had a methane mole fraction of xmethane,grav = (0.506875 ± 0.00019) and a propane mole fraction of xpropane,grav = (0.493125 ± 0.00019). NMR samples were prepared in two types of commercially available sample tubes that seal with a PTFE piston. Sample pressures ranged from 0.02 to 0.5 MPa. An analysis of measurement uncertainty for the NMR method resulted in combined standard uncertainties that decreased from 0.0082 x to 0.0010 x, as the pressure increased from 0.02 to 0.5 MPa. The larger uncertainties at lower pressures were primarily caused by uncertainties associated with phasing and baseline correction. A key difficulty in working with gas-phase samples, especially at lower pressures, is that the spectral peaks are inherently broad. Consequently, peak overlap was problematic, and it was not always possible to integrate a high percentage of a peak's intensity. However, with corrections to the integrated areas, based on the assumption of ideal Lorentzian peak shapes, excellent agreement between the NMR analyses and the gravimetric composition was observed across the entire pressure range. These experiments demonstrate the potential of 1H NMR for quantitative composition determinations of low-pressure gas-phase mixtures.

Metrologically traceable quantification of trifluoroacetic acid content in peptide reference materials by 19F solid-state NMR

Although solution-state NMR is frequently used in metrologically-traceable quantification studies, this is not the case for solid-state NMR. However, solid-state NMR allows quantification of substances without the need of dissolution, providing a truly non-destructive approach, and extending metrologically-traceable quantitative NMR to sample classes that are difficult to characterize in solution. In this contribution we present a thorough and rigorous protocol for 19F quantitative solid-state NMR employing a certified reference material as external calibrant to provide metrological traceability to absolutely quantify the content of trifluoroacetic acid (TFA) in a peptide sample, typically the major impurity in synthetic peptides. The protocol includes determining the quantitative volume of the solid-state NMR sample holder (rotor), the ERETIC (electronic reference to access in vivo concentrations) method (Akoka et al 1999 Anal. Chem. 71 2554) to compensate for variations in the sensitivity of the radio frequency resonant circuit when an external calibrant is used, and the EASY (elimination of artefacts in NMR spectroscopy) method (Jaeger and Hemmann 2014 Solid State Nucl. Magn. Reson. 57–58 22) to effectively suppress the 19F NMR background signal from the probe head. We applied the protocol to quantify the amount of TFA in a candidate NRC certified reference material of the peptide angiotensin II. The results obtained by 19F quantitative solid-state NMR are in excellent agreement with those obtained by quantitative NMR in solution employing an internal calibrant.