NMR spectroscopy reveals unexpected structural variation at the protein–protein interface in MHC class I molecules

It is well established that non-uniform sampling (NUS) allows acquisition of multi-dimensional NMR spectra at a resolution that cannot be obtained with traditional uniform acquisition through the indirect dimensions. However, the impact of NUS on the signal-to-noise ratio (SNR) and sensitivity are less well documented. SNR and sensitivity are essential aspects of NMR experiments as they define the quality and extent of data that can be obtained. This is particularly important for spectroscopy with low concentration samples of biological macromolecules. There are different ways of defining the SNR depending on how to measure the noise, and the distinction between SNR and sensitivity is often not clear. While there are defined procedures for measuring sensitivity with high concentration NMR standards, such as sucrose, there is no clear or generally accepted definition of sensitivity when comparing different acquisition and processing methods for spectra of biological macromolecules with many weak signals close to the level of noise. Here we propose tools for estimating the SNR and sensitivity of NUS spectra with respect to sampling schedule and reconstruction method. We compare uniformly acquired spectra with NUS spectra obtained in the same total measuring time. The time saving obtained when only 1/k of the Nyquist grid points are sampled is used to measure k-fold more scans per increment. We show that judiciously chosen NUS schedules together with suitable reconstruction methods can yield a significant increase of the SNR within the same total measurement time. Furthermore, we propose to define the sensitivity as the probability to detect weak peaks and show that time-equivalent NUS can considerably increase this detection sensitivity. The sensitivity gain increases with the number of NUS indirect dimensions. Thus, well-chosen NUS schedules and reconstruction methods can significantly increase the information content of multidimensional NMR spectra of challenging biological macromolecules.     

Optimization of 13C direct detection NMR methods

(13)C-detected experiments are still limited by their inherently lower sensitivity, as compared to the equivalent (1)H-detected experiments. Improving the sensitivity of (13)C detection methods remains a significant area of NMR research that may provide better means for studying large macromolecular systems by NMR. In this communication, we show that (13)C-detected experiments are less sensitive to the salt concentration of the sample solution than (1)H-detected experiments. In addition, acquisition can be started with anti-phase coherence, resulting in higher sensitivity due to the elimination of the final INEPT transfer step.     

Hyperpolarized NMR metabolomics

Hyperpolarized NMR is a promising approach to address the sensitivity limits of conventional NMR metabolomics approaches, which currently fails to detect minute metabolite concentrations in biological samples. This review describes how tremendous signal enhancement offered by dissolution-dynamic nuclear polarization and parahydrogen-based techniques can be fully exploited for molecular omics sciences. Recent developments, including the combination of hyperpolarization techniques with fast multi-dimensional NMR implementation and quantitative workflows are described, and a comprehensive comparison of existing hyperpolarization techniques is proposed. High-throughput, sensitivity, resolution and other relevant challenges that should be tackled for a general application of hyperpolarized NMR in metabolomics are discussed.     

NMR of live systems

Continuous non-destructive in vivo biochemical measurements have been a desired but seemingly unattainable goal for many years. Now, however, the use of nuclear magnetic resonance (NMR) seems to be close to reaching that end. This review can be divided into two sections. The first introduces and discusses relevant NMR parameters such as chemical shift and relaxation times form the view of their application to biologic systems. The second part highlights recent achievements by showing the variety of chores NMR can perform. In particular, the topics of pH measurement, quantitation and identification of phosphorus and carbon metabolites, and enzyme kinetics are all discussed. Also mentioned are limitations of NMR such as low sensitivity. Finally, there is a discussion of the new NMR technologies such as whole body proton imaging and topical NMR.     

Photo-CIDNP NMR methods for studying protein folding

Chemically induced dynamic nuclear polarization (CIDNP) is a nuclear magnetic resonance phenomenon that can be used to probe the solvent-accessibility of tryptophan, tyrosine, and histidine residues in proteins by means of laser-induced photochemical reactions, resulting in significant enhancement of NMR signals. CIDNP offers good sensitivity as a surface probe of protein structure and is particularly powerful in time-resolved NMR measurements. Real-time, rapid-injection protein refolding experiments permit the observation of changes in the accessibility of specific residues during the folding process. CIDNP pulse-labeling gives information on the accessibility of residues in partially structured proteins (e.g., molten globule states) whose NMR spectra are broad and poorly resolved. Heteronuclear two-dimensional (15)N-(1)H CIDNP techniques allow identification of surface-accessible residues with improved resolution and sensitivity. These methods offer residue-specific structural and kinetic information on transient folding intermediates and other partially folded states of proteins that are not readily available from more routine NMR techniques.     

Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy

Xenon-binding sites in proteins have led to a number of applications of xenon in biochemical and structural studies. Here we further develop the utility of 129Xe NMR in characterizing specific xenon-protein interactions. The sensitivity of the 129Xe chemical shift to its local environment and the intense signals attainable by optical pumping make xenon a useful NMR reporter of its own interactions with proteins. A method for detecting specific xenon-binding interactions by analysis of 129Xe chemical shift data is illustrated using the maltose binding protein (MBP) from Escherichia coli as an example. The crystal structure of MBP in the presence of 8atm of xenon confirms the binding site determined from NMR data. Changes in the structure of the xenon-binding cavity upon the binding of maltose by the protein can account for the sensitivity of the 129Xe chemical shift to MBP conformation. 129Xe NMR data for xenon in solution with a number of cavity containing phage T4 lysozyme mutants show that xenon can report on cavity structure. In particular, a correlation exists between cavity size and the binding-induced 129Xe chemical shift. Further applications of 129Xe NMR to biochemical assays, including the screening of proteins for xenon binding for crystallography are considered.     

Al NMR: a novel NMR data processing program optimized for sparse sampling

Sparse sampling in biomolecular multidimensional NMR offers increased acquisition speed and resolution and, if appropriate conditions are met, an increase in sensitivity. Sparse sampling of indirectly detected time domains combined with the direct truly multidimensional Fourier transform has elicited particular attention because of the ability to generate a final spectrum amenable to traditional analysis techniques. A number of sparse sampling schemes have been described including radial sampling, random sampling, concentric sampling and variations thereof. A fundamental feature of these sampling schemes is that the resulting time domain data array is not amenable to traditional Fourier transform based processing and phasing correction techniques. In addition, radial sampling approaches offer a number of advantages and capabilities that are also not accessible using standard NMR processing techniques. These include sensitivity enhancement, sub-matrix processing and determination of minimal sets of sampling angles. Here we describe a new software package (Al NMR) that enables these capabilities in the context of a general NMR data processing environment.     

The application of micro-coil NMR probe technology to metabolomics of urine and serum

Increasing the sensitivity and throughput of NMR-based metabolomics is critical for the continued growth of this field. In this paper the application of micro-coil NMR probe technology was evaluated for this purpose. The most commonly used biofluids in metabolomics are urine and serum. In this study we examine different sample limited conditions and compare the detection sensitivity of the micro-coil with a standard 5?mm NMR probe. Sample concentration is evaluated as a means to leverage the greatly improved mass sensitivity of the micro-coil probes. With very small sample volumes, the sensitivity of the micro-coil probe does indeed provide a significant advantage over the standard probe. Concentrating the samples does improve the signal detection, but the benefits do not follow the expected linear increase and are both matrix and metabolite specific. Absolute quantitation will be affected by concentration, but an analysis of relative concentrations is still possible. The choice of the micro-coil probe over a standard tube based probe will depend upon a number of factors including number of samples and initial volume but this study demonstrates the feasibility of high-throughput metabolomics with the micro-probe platform.     

Detection of site-specific binding and co-binding of ligands to macromolecules using 19F NMR

Study of ligand-macromolecular interactions by 19F nuclear magnetic resonance (NMR) spectroscopy affords many opportunities for obtaining molecular biochemical and pharmaceutical information. This is due to the absence of a background fluorine signal, as well as the relatively high sensitivity of 19F NMR. Use of fluorine-labeled ligands enables one to probe not only binding and co-binding phenomena to macromolecules, but also can provide data on binding constants, stoichiometries, kinetics, and conformational properties of these complexes. Under conditions of slow exchange and macromolecule-induced chemical shifts, multiple 19F NMR resonances can be observed for free and bound ligands. These shifted resonances are a direct correlate of the concentration of ligand bound in a specific state rather than the global concentrations of bound or free ligand which are usually determined using other techniques such as absorption spectroscopy or equilibrium dialysis. Examples of these interactions are demonstrated both from the literature and from interactions of 5-fluorotryptophan, 5-fluorosalicylic acid, flurbiprofen, and sulindac sulfide with human serum albumin. Other applications of 19F NMR to study of these interactions in vivo, as well for receptor binding and metabolic tracing of fluorinated drugs and proteins are discussed.     

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.     

A non-uniform sampling approach enables studies of dilute and unstable proteins

NMR spectroscopy is a powerful method in structural and functional analysis of macromolecules and has become particularly prevalent in studies of protein structure, function and dynamics. Unique to NMR spectroscopy is the relatively low constraints on sample preparation and the high level of control of sample conditions. Proteins can be studied in a wide range of buffer conditions, e.g. different pHs and variable temperatures, allowing studies of proteins under conditions that are closer to their native environment compared to other structural methods such as X-ray crystallography and electron microscopy. The key disadvantage of NMR is the relatively low sensitivity of the method, requiring either concentrated samples or very lengthy data-acquisition times. Thus, proteins that are unstable or can only be studied in dilute solutions are often considered practically unfeasible for NMR studies. Here, we describe a general method, where non-uniform sampling (NUS) allows for signal averaging to be monitored in an iterative manner, enabling efficient use of spectrometer time, ultimately leading to savings in costs associated with instrument and isotope-labelled protein use. The method requires preparation of multiple aliquots of the protein sample that are flash-frozen and thawed just before acquisition of a short NMR experiments carried out while the protein is stable (12 h in the presented case). Non-uniform sampling enables sufficient resolution to be acquired for each short experiment. Identical NMR datasets are acquired and sensitivity is monitored after each co-added spectrum is reconstructed. The procedure is repeated until sufficient signal-to-noise is obtained. We discuss how maximum entropy reconstruction is used to process the data, and propose a variation on the previously described method of automated parameter selection. We conclude that combining NUS with iterative co-addition is a general approach, and particularly powerful when applied to unstable proteins.     

CP-MAS and Solution NMR Studies of Allosteric Communication in CA-assemblies of HIV-1

Solution and solid-state NMR spectroscopy are highly complementary techniques for studying structure and dynamics in very high molecular weight systems. Here we have analysed the dynamics of HIV-1 capsid (CA) assemblies in presence of the cofactors IP6 and ATPγS and the host-factor CPSF6 using a combination of solution state and cross polarisation magic angle spinning (CP-MAS) solid-state NMR. In particular, dynamical effects on ns to µs and µs to ms timescales are observed revealing diverse motions in assembled CA.Using CP-MAS NMR, we exploited the sensitivity of the amide/Cα-Cβ backbone chemical shifts in DARR and NCA spectra to observe the plasticity of the HIV-1 CA tubular assemblies and also map the binding of cofactors and the dynamics of cofactor-CA complexes. In solution, we measured how the addition of host- and co-factors to CA -hexamers perturbed the chemical shifts and relaxation properties of CA-Ile and -Met methyl groups using transverse-relaxation-optimized NMR spectroscopy to exploit the sensitivity of methyl groups as probes in high-molecular weight proteins. These data show how dynamics of the CA protein assembly over a range of spatial and temporal scales play a critical role in CA function. Moreover, we show that binding of IP6, ATPγS and CPSF6 results in local chemical shift as well as dynamic changes for a significant, contiguous portion of CA, highlighting how allosteric pathways communicate ligand interactions between adjacent CA protomers.     

Selectively 2H-labeled Glu/Asp: application to pKa measurements in Abeta amyloid peptides.

Human Abeta peptides have been linked to Alzheimer's disease, and it is hypothesized that formation of amyloid as well as neurotoxicity are important events in the etiology of the disease. Previous studies have shown that the soluble precursor to Alzheimer's amyloid undergoes a pH-dependent folding transition as the self-assembly activity appears, and based upon inter-residue proximities, it was suspected that stabilization of the soluble form might rely upon formation of an intramolecular salt-bridge. However, pKa studies on a model 17-residue Abeta fragment supported an electrostatic model where a solvation imperative for charged side-chain atoms drives the folding process. To explore this model in an active 26-residue fragment as well as the full-length 40-residue Abeta peptide, pKa measurements were performed via 1H and 2H NMR. To overcome issues related to sensitivity and spin system degeneracy, specifically deuterated allyl protected-Fmoc amino acids were synthesized for incorporation into a series of peptides, and a high sensitivity 2H observe NMR probe was constructed.     

Applications of NMR in drug discovery

NMR, already some 50 years old, has long been an invaluable analytical method in industry for verification of chemical synthesis and compound characterisation. The range of molecular information accessible through NMR, however, offers a far larger horizon of applications. Of these, ligand screening by NMR has emerged as a very promising new method in drug discovery. Its unmatched screening sensitivity, combined with the abundance of available information on the structure and nature of molecular binding, justifies the growing interest in this dynamically expanding NMR application.     

Perfluoro‐tert‐butyl‐homoserine as a sensitive 19F NMR reporter for peptide–membrane interactions in solution

Fluorine (¹⁹F) NMR is a valuable tool for studying dynamic biological processes. However, increasing the sensitivity of fluorinated reporter molecules is a key to reducing acquisition times and accessing transient biological interactions. Here, we evaluate the utility a novel amino acid, l ‐O‐(perfluoro‐t‐butyl)‐homoserine (pFtBSer), that can easily be synthesized and incorporated into peptides and provides greatly enhanced sensitivity over currently used ¹⁹F biomolecular NMR probes. Incorporation of pFtBSer into the potent antimicrobial peptide MSI‐78 results in a sharp ¹⁹F NMR singlet that can be readily detected at concentrations of 5 µm and lower. We demonstrate that pFtBSer incorporation into MSI‐78 provides a sensitive tool to study binding through ¹⁹F NMR chemical shift and nuclear relaxation changes. These results establish future potential for pFtBSer to be incorporated into various proteins where NMR signal sensitivity is paramount, such as in‐cell investigations. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Observation of slow dynamic exchange processes in Ras protein crystals by 31P solid state NMR spectroscopy

The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3mm(3) size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10(15) phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras-nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras-GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal.     

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.     

Metabolomic analysis using optimized NMR and statistical methods

NMR-based metabolomics requires robust automated methodologies, and the accuracy of NMR-based metabolomics data is greatly influenced by the reproducibility of data acquisition and processing methods. Effective water resonance signal suppression and reproducible spectral phasing and baseline traces across series of related samples are crucial for statistical analysis. We assess robustness, repeatability, sensitivity, selectivity, and practicality of commonly used solvent peak suppression methods in the NMR analysis of biofluids with respect to the automated processing of the NMR spectra and the impact of pulse sequence and data processing methods on the sensitivity of pattern recognition and statistical analysis of the metabolite profiles. We introduce two modifications to the excitation sculpting pulse sequence whereby the excitation solvent suppression pulse cascade is preceded by low-power water resonance presaturation pulses during the relaxation delay. Our analysis indicates that combining water presaturation with excitation sculpting water suppression delivers the most reproducible and information-rich NMR spectra of biofluids.     

Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear ¹³C and ¹⁵N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes.     

31P-NMR study of natural phytoplankton samples

A new fixation method was developed for the Nuclear Magnetic Resonance (NMR) study of natural phytoplankton samples collected in situ. To test NMR reliability, a Chlorella continuous culture was used in a phosphorus deficiency recovery experiment. The method was then applied to natural metalimnetic cyanobacterial plankton. The maximum Entropy Method was used to enhance the generally poor signal to noise ratio resulting from the low amount of available material and NMR sensitivity. Suggestions are made on how to improve reliability.