Nuclear Magnetic Resonance Sample Research Articles

Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance(NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the β subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 μM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

The combination of inorganic phosphate and pyrophosphate 31 P-NMR for the electrodeless pH determination in the 5-12 range.

Potentiometry is the primary pH measurement method, but alternatives are sought beyond glass electrodes operative limitations. In nuclear magnetic resonance (NMR) experiments, electrodeless pH sensing is important to track changes along titrations, during chemical reactions or inside compartmentalized environments inaccessible to electrodes, for instance. Although several interesting NMR pH indicators have been already presented, the potential of inorganic phosphate is overlooked, despite its common presence in NMR samples as the buffer main component. Its use for electrodeless pH determination can be expanded by exploiting all its three proton dissociations. This study was aimed at verifying the use of inorganic phosphate 31 P chemical shift to sense pH variations, and at exploring the complementary use of pyrophosphate ions to cover a wide pH range. A simple set of equations is presented to utilize both phosphate and pyrophosphate 31 P chemical shift in combination for accurate pH determination without a glass electrode over the 5-12 pH range, and without affecting the spectrum of other nuclei. The present study demonstrated an average deviation of 0.09 (maximum <0.2) pH unit from glass electrode measurements. The trimethylphosphate can be used as a suitable chemical shift reference for both 31 P and 1 H (also 13 C), with its hydrolysis being significant only at pH > 12. The method was also demonstrated by determining the pKa of three distinct molecules in a mixture and by comparing the results to those obtained when the glass electrode was used to measure the pH. The approach shown here can be easily tuned to different experimental conditions.

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies.

The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10-45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

Solution-state nuclear magnetic resonance spectroscopy of crystalline cellulosic materials using a direct dissolution ionic liquid electrolyte.

Owing to its high sustainable production capacity, cellulose represents a valuable feedstock for the development of more sustainable alternatives to currently used fossil fuel-based materials. Chemical analysis of cellulose remains challenging, and analytical techniques have not advanced as fast as the development of the proposed materials science applications. Crystalline cellulosic materials are insoluble in most solvents, which restricts direct analytical techniques to lower-resolution solid-state spectroscopy, destructive indirect procedures or to 'old-school' derivatization protocols. While investigating their use for biomass valorization, tetralkylphosphonium ionic liquids (ILs) exhibited advantageous properties for direct solution-state nuclear magnetic resonance (NMR) analysis of crystalline cellulose. After screening and optimization, the IL tetra-n-butylphosphonium acetate [P4444][OAc], diluted with dimethyl sulfoxide-d6, was found to be the most promising partly deuterated solvent system for high-resolution solution-state NMR. The solvent system has been used for the measurement of both 1D and 2D experiments for a wide substrate scope, with excellent spectral quality and signal-to-noise, all with modest collection times. The procedure initially describes the scalable syntheses of an IL, in 24-72 h, of sufficient purity, yielding a stock electrolyte solution. The dissolution of cellulosic materials and preparation of NMR samples is presented, with pretreatment, concentration and dissolution time recommendations for different sample types. Also included is a set of recommended 1D and 2D NMR experiments with parameters optimized for an in-depth structural characterization of cellulosic materials. The time required for full characterization varies between a few hours and several days.

Reconversion of Parahydrogen Gas in Surfactant-Coated Glass NMR Tubes.

The application of parahydrogen gas to enhance the magnetic resonance signals of a diversity of chemical species has increased substantially in the last decade. Parahydrogen is prepared by lowering the temperature of hydrogen gas in the presence of a catalyst; this enriches the para spin isomer beyond its normal abundance of 25% at thermal equilibrium. Indeed, parahydrogen fractions that approach unity can be attained at sufficiently low temperatures. Once enriched, the gas will revert to its normal isomeric ratio over the course of hours or days, depending on the surface chemistry of the storage container. Although parahydrogen enjoys long lifetimes when stored in aluminum cylinders, the reconversion rate is significantly faster in glass containers due to the prevalence of paramagnetic impurities that are present within the glass. This accelerated reconversion is especially relevant for nuclear magnetic resonance (NMR) applications due to the use of glass sample tubes. The work presented here investigates how the parahydrogen reconversion rate is affected by surfactant coatings on the inside surface of valved borosilicate glass NMR sample tubes. Raman spectroscopy was used to monitor changes to the ratio of the (J: 0 → 2) vs. (J: 1 → 3) transitions that are indicative of the para and ortho spin isomers, respectively. Nine different silane and siloxane-based surfactants of varying size and branching structures were examined, and most increased the parahydrogen reconversion time by 1.5×-2× compared with equivalent sample tubes that were not treated with surfactant. This includes expanding the pH2 reconversion time from 280 min in a control sample to 625 min when the same tube is coated with (3-Glycidoxypropyl)trimethoxysilane.

Theoretical analysis of flow effects in spatially encoded diffusion NMR.

The measurement of translational diffusion coefficients by nuclear magnetic resonance (NMR) spectroscopy is essential in a broad range of fields, including organic, inorganic, polymer, and supramolecular chemistry. It is also a powerful method for mixture analysis. Spatially encoded diffusion NMR (SPEN DNMR)" is a time efficient technique to collect diffusion NMR data, which is particularly relevant for the analysis of samples that evolve in time. In many cases, motion other than diffusion is present in NMR samples. This is, for example, the case of flow NMR experiments, such as in online reaction monitoring and in the presence of sample convection. Such motion is deleterious for the accuracy of DNMR experiments in general and for SPEN DNMR in particular. Limited theoretical understanding of flow effects in SPEN DNMR experiments is an obstacle for their broader experimental implementation. Here, we present a detailed theoretical analysis of flow effects in SPEN DNMR and of their compensation, throughout the relevant pulse sequences. This analysis is validated by comparison with numerical simulation performed with the Fokker-Planck formalism. We then consider, through numerical simulation, the specific cases of constant, laminar, and convection flow and the accuracy of SPEN DNMR experiments in these contexts. This analysis will be useful for the design and implementation of fast diffusion NMR experiments and for their applications.

Sample illumination device facilitates in situ light-coupled NMR spectroscopy without fibre optics

In situ illumination of liquid-state nuclear magnetic resonance (NMR) samples makes it possible for a wide range of light-dependent chemical and biological phenomena to be studied by the powerful analytical technique. However, the position of an NMR sample deep within the bore of the spectrometer magnet renders such illumination challenging. Here, we demonstrate the working principles of a sample illumination device (NMRtorch) where a lighthead containing an LED array is positioned directly at the top of an NMRtorch tube which is inserted into the NMR spectrometer. The wall of the tube itself acts as a light guide, illuminating the sample from the outside. We explore how this new setup performs in a number of photo-NMR applications, including photoisomerisation and photo-chemically induced dynamic nuclear polarisation (photo-CIDNP), and demonstrate the potential for ultraviolet (UV) degradation studies with continuous online NMR assessment. This setup enables users of any typical liquid-state spectrometer to easily perform in situ photo-NMR experiments, using a wide range of wavelengths.

An Easy and Reliable Method for the Mitigation of Deuterated Chloroform Decomposition to Stabilise Susceptible NMR Samples

Highly reactive decomposition products of deuterated chloroform can deteriorate samples dissolved in this commonly used solvent for nuclear magnetic resonance (NMR) spectroscopy. Samples for metabolomics studies often contain a complex mixture of sensitive substances such as phospholipids, peptides, unsaturated fatty acids or vitamins. If these react with decomposition products (of chloroform), abnormal NMR spectra could result, e.g., signal shifts depending on pH, attenuation of signals over time due to chemical changes of analytes or new signals from reaction products. Such irreproducibly influenced spectra are especially problematic for non-targeted analysis methods using automated chemometrical data evaluation. To prevent these artefacts, chlorine, phosgene and hydrochloric acid need to be eliminated from deuterated chloroform before its use. Since the common stabilisation methods have proven to be insufficient for sensitive NMR samples, another purging method has been tested: Mitigation is easily and reliably achieved by washing the deuterated chloroform with concentrated disodium carbonate solution and subsequent desiccation with oven-dried disodium carbonate.

Analytical Validation of GFRNMR: A Blood-Based Multiple Biomarker Assay for Accurate Estimation of Glomerular Filtration Rate

Accurate and precise monitoring of kidney function is critical for a timely and reliable diagnosis of chronic kidney disease (CKD). The determination of kidney function usually involves the estimation of the glomerular filtration rate (eGFR). We recently reported the clinical performance of a new eGFR equation (GFRNMR) based on the nuclear magnetic resonance (NMR) measurement of serum myo-inositol, valine, and creatinine, in addition to the immunoturbidometric quantification of serum cystatin C, age and sex. We now describe the analytical performance evaluation of GFRNMR according to the Clinical and Laboratory Standards Institute guidelines. Within-laboratory coefficients of variation (CV%) of the GFRNMR equation did not exceed 4.3%, with a maximum CV% for repeatability of 3.7%. Between-site reproducibility (three sites) demonstrated a maximum CV% of 5.9%. GFRNMR stability was demonstrated for sera stored for up to 8 days at 2–10°C and for NMR samples stored for up to 10 days in the NMR device at 6 ± 2°C. Substance interference was limited to 4/40 (10.0%) of the investigated substances, resulting in an underestimated GFRNMR (for glucose and metformin) or a loss of results (for naproxen and ribavirin) for concentrations twice as high as usual clinical doses. The analytical performances of GFRNMR, combined with its previously reported clinical performance, support the potential integration of this NMR method into clinical practice.

Noise model of the cryogenic nuclear magnetic resonance spectroscopy system's receiving chain

One of the challenges in modern nuclear magnetic resonance (NMR) is achieving its highest possible measuring sensitivity. This is because modern NMR samples' response signals decrease, so background noise of the used NMR spectroscopy system causes bigger problems. As a solution, both software and hardware interventions were applied. However, these improvements were obtained experimentally, so the reason for their proper operation and upper limit is usually unknown. Recently, a noise model of the NMR spectroscopy system, which shows both the reason for proper operation and the upper limit of the applied improvements, was introduced. A Javascript-based calculator, based on the introduced model, has been developed and made available online as a user-friendly website that can be run on the most commonly used Internet browsers. To the authors' knowledge, this calculator is the first of its kind that analyses noise properties in NMR. Using it, one can a priori make both sensitivity prediction of practical NMR systems in physics and material science and quantitative analysis of its noise properties. Consequently, overall measurement duration can be shortened down to one half of the current duration. This is an immense improvement, as some modern NMR measurements consume more than ten hours per measurement.

Simple and Rapid (Extraction) Protocol for NMR Metabolomics-Direct Measurement of Hydrophilic and Hydrophobic Metabolites Using Slice Selection.

Investigating the metabolic profiles of solid sample materials with solution nuclear magnetic resonance (NMR) spectroscopy requires the extraction of these metabolites. This is commonly done by using two immiscible solvents such as water and chloroform for extraction. Subsequent solvent removal makes these extraction procedures very time-consuming. To shorten the preparation time of the NMR sample, the following protocol is proposed: the metabolites from a solid or liquid sample are extracted directly in the NMR tube, the NMR tube is centrifuged, and the metabolite profiles in the aqueous and organic phases are determined by using slice-selective proton NMR experiments. This protocol was tested with 11 black teas and 11 green teas, which can be easily distinguished by their metabolic profiles in the aqueous phase. As a test case for liquid samples, 29 milk samples were investigated. The geographical origin of the diaries where the milk was processed could not be determined unequivocally from the metabolic profiles of the hydrophilic metabolites; however, this was easily seen in the lipid profiles. As shown for the different test samples, the extraction protocol in combination with slice-selection NMR experiments is suitable for metabolic investigations. Because samples are rapidly processed, this approach can be used to explore different extraction strategies for metabolite isolation.

Development and Performance Evaluation of Solid-Free Drilling Fluid for CBM Reservoir Drilling in Central Hunan

Solid-free drilling fluid is a matter of cardinal significance in the course of Coal bed Methane (CBM) reservoir drilling. This study evaluated the performance of solid-free CBM drilling fluid in central Hunan. Three types of surfactants, namely TX-10 (nonionic), HSB1618 (zwitterionic) and penetrant T (anionic), were added in basic fluid at various concentrations of 0.05, 0.10 and 0.15% (m/m). This study comprised of drilling fluid rheology, sample mineral analysis, sample nuclear magnetic resonance (NMR) scanning, sample wettability, and sample surface micro characteristics tests. The results show that TX-10 and HSB1618 enhance the rheological properties of drilling fluid, such as yield point and gel strength. Penetrant T has opposite effect on it. It was found that the minimum American Petroleum Institute (API) filtration is only 0.3 mL. This study adopted a new method using laser particle size analyzer to evaluate suspension performance. Based on the surface micro characteristics of the sample and the NMR scanning tests, it is found that the residual amount of basic fluid + HSB1618 in the sample is the smallest. The wettability modification curve indicates that three surfactants decrease the sample’s hydrophobicity. With the increase of surfactant concentration, all above parameters change regularly. The basic fluid + 0.10% HSB1618 has the strongest hydrophobicity for sample at pH = 10. This study obtained a set of solid-free drilling fluid system, which provides better suspension capacity and large contact angle and reduces residue of drilling fluid in CBM reservoir. Ultimately, it can accelerate the desorption of coal gas and reduce damage to the reservoir.

Water-Soluble Glycosylated Resveratrol for Measuring Residual Dipolar Coupling in NMR

Residual dipolar coupling (RDC) is a newly emerging technique for obtaining more precise conformational features of molecules in nuclear magnetic resonance (NMR), which can be observed when the molecules are oriented in NMR sample solutions. Unlike the case of solubilization of resveratrol (Res) in aqueous media by surfactants, multiglycosylated resveratrol (GlyRes) was dispersed in aqueous media forming nanoparticles with an extremely small size. In the presence of an NMR alignment medium of bacteriophage, GlyRes molecules were efficiently oriented in the strong magnetic field allowing for observations of RDCs in NMR. Structural refinement using the observed RDC values enabled differentiation of trans and cis forms of Res.

Solution structure and oligomeric state of the E. coliglycerol facilitator

Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.

Characterization of Volcanic Reservoirs Requires Comprehensive NMR Tests

This article, written by JPT Technology Editor Judy Feder, contains highlights of paper IPTC 19378, “Comprehensive Evaluation of NMR Characteristics of Complex Volcanic Reservoirs With Different Types of Rock Lithology,” by Junchang Sun, Shijie Zhang, Jieming Wang, Hekun Guo, Chun Li, Hongcheng Xu, Sinan Zhu, and Kai Zhao, PetroChina, prepared for the 2019 International Petroleum Technology Conference, Beijing, 26–28 March. The paper has not been peer reviewed. Copyright 2019 International Petroleum Technology Conference. Reproduced by permission. This paper discusses a study undertaken to gain better understanding of nuclear magnetic resonance (NMR) characteristics of volcanic reservoirs with different lithologies. Results of the study confirmed that accurate identification of reservoir-interval lithology, achieved through comprehensive laboratory NMR investigation of the volcanic rock, is a primary prerequisite of correct interpretation of NMR logging of volcanic reservoirs. The research results play an important role in guiding in-depth understanding of NMR characteristics of volcanic reservoirs and the implementation of NMR reservoir evaluation in corresponding areas. Introduction The introduction of NMR to the petroleum industry in the 1990s led to a breakthrough in logging. NMR can distinguish between movable and irreducible fluid, and is currently the only logging technology that can determine reservoir permeability. When used to interpret and evaluate complex reservoirs such as volcanic rocks and low-resistivity oil and gas layers, NMR provides logging analysts with new ideas and techniques to deal with various difficulties. By analyzing the NMR relaxation-time (T2) spectrum of rock plugs at a fully brine-saturated state, the distribution of reservoir pore size can be evaluated, and important reservoir evaluation parameters, including effective porosity, NMR permeability, and movable fluid saturation, can be obtained. Numerous studies have shown that, compared with sandstones and carbonates, volcanic reservoirs are much more complex and heterogeneous because of the special eruption diagenesis mechanism, many types of rock lithology, various mineral compositions, and a broad range of pore sizes. The models currently used for NMR logging interpretation are suitable only for sedimentary reservoirs. Consequently, accurate characterization of volcanic reservoirs using advanced NMR logging requires a comprehensive laboratory NMR investigation of volcanic rock. Materials and Methods For the study described in the complete paper, NMR sample analysis technology was adopted to perform NMR measurements and other related tests on a total of 108 low-permeability rock plugs from the Xushen reservoir of the Daqing field, the Changling reservoir of the Jilin field, and the Dixi reservoir of the Xinjiang volcanic gas field. The rock plugs comprised nine types of lithology representing the main producing formation lithology from the three reservoirs. Tests included CT scans, thin-section petrography, mercury injection, and mineral-composition analysis. Centrifuge tests were conducted with maximum centrifugal forces up to 500 psi to explore the suitable capillary pressure for T2cutoff determination. The paper includes a step-by-step description of the experimental procedure.

A Quick and Colorful Method to Measure Low-Level Contaminations of Paramagnetic Ni2+ in Protein Samples Purified by Immobilized Metal Ion Affinity Chromatography.

Isotopic labeling of recombinantly expressed proteins is generally required for investigation by modern nuclear magnetic resonance (NMR) methods. Purification strategies of the labeled proteins often include the use of a polyhistidine affinity tag (His-tag) and immobilized metal ion affinity chromatography (IMAC). Described herein are rapid and inexpensive qualitative and quantitative assays to determine the concentration of paramagnetic Ni2+ in protein samples purified by IMAC. Both qualitative and quantitative colorimetric methods detect the amount of Ni2+ via the color change produced when a [Ni(PAR)n]2+ (PAR=4-(2-pyridylazo)resorcinol, n=1, 2) complex is formed. The qualitative assay provides a rapid visual test for the presence of Ni2+ in the low micromolar range in a sample of interest. The usefulness of the spectroscopic quantitative assay is illustrated by: (i) detecting a 12μM Ni2+ contamination in an NMR sample containing 950μM of the 7.5kDa α3W protein purified by a standard His-tag Ni2+/IMAC approach and (ii) showing that the 15N-HSQC spectrum of the α3W NMR sample, containing 1 paramagnetic Ni2+ ion per 80 protein molecules, displays clear line broadening of both water and protein spectral lines. We also (iii) measured Ni2+ release during the equilibration, wash, and elution steps of three commonly used Ni2+/IMAC resins when following manufacturer's protocols. The concentration of Ni2+ detected in elutes of the three resins ranged from 2μM to nearly 1mM.

Diffusion in Nanoporous Materials: Novel Insights by Combining MAS and PFG NMR

Pulsed field gradient (PFG) nuclear magnetic resonance (NMR) allows recording of molecular diffusion paths (notably, the probability distribution of molecular displacements over typically micrometers, covered during an observation time of typically milliseconds) and has thus proven to serve as a most versatile means for the in-depth study of mass transfer in complex materials. This is particularly true with nanoporous host materials, where PFG NMR enabled the first direct measurement of intracrystalline diffusivities of guest molecules. Spatial resolution, i.e., the minimum diffusion path length experimentally observable, is limited by the time interval over which the pulsed field gradients may be applied. In “conventional” PFG NMR measurements, this time interval is determined by a characteristic quantity of the host-guest system under study, the so-called transverse nuclear magnetic relaxation time. This leads, notably when considering systems with low molecular mobilities, to severe restrictions in the applicability of PFG NMR. These restrictions may partially be released by performing PFG NMR measurements in combination with “magic-angle spinning” (MAS) of the NMR sample tube. The present review introduces the fundamentals of this technique and illustrates, via a number of recent cases, the gain in information thus attainable. Examples include diffusion measurements with nanoporous host-guest systems of low intrinsic mobility and selective diffusion measurement in multicomponent systems.

Current NMR Techniques for Structure-Based Drug Discovery.

A variety of nuclear magnetic resonance (NMR) applications have been developed for structure-based drug discovery (SBDD). NMR provides many advantages over other methods, such as the ability to directly observe chemical compounds and target biomolecules, and to be used for ligand-based and protein-based approaches. NMR can also provide important information about the interactions in a protein-ligand complex, such as structure, dynamics, and affinity, even when the interaction is too weak to be detected by ELISA or fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) or to be crystalized. In this study, we reviewed current NMR techniques. We focused on recent progress in NMR measurement and sample preparation techniques that have expanded the potential of NMR-based SBDD, such as fluorine NMR (19F-NMR) screening, structure modeling of weak complexes, and site-specific isotope labeling of challenging targets.

Augmenting microwave irradiation in MAS DNP NMR samples at 263 GHz

The magnetic microwave field strength and its detailed spatial distribution in magic-angle spinning (MAS) nuclear magnetic resonance (NMR) probes capable of dynamic nuclear polarization (DNP) is investigated by numerical simulations with the objective to augment the magnetic microwave amplitude by structuring the sample in the mm and sub-mm range and by improving the coupling of the incident microwave beam to the sample. As it will be shown experimentally, both measures lead to an increase of the microwave efficiency in DNP MAS NMR.

Analysis of Prion Protein Structure Using Nuclear Magnetic Resonance Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful experimental tool for obtaining information on three-dimensional (3D) structures of proteins at atomic resolution. In inherited forms of prion diseases, misfolding of cellular prion protein, PrPC, into its pathological form, PrPSc, is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest stages of the conformational changes leading to spontaneous generation of prions in inherited forms of prion diseases may benefit from detailed structural analysis of different human (Hu) PrP variants. Here, we describe the protocol for structure determination of HuPrP variants by NMR spectroscopy in solution that consists of preparation of NMR samples, acquisition of NMR data, NMR resonance assignments, and structure calculation.