Cerebral phosphoester signals measured by 31P magnetic resonance spectroscopy at 3 and 7 Tesla.

Abnormal cell membrane metabolism is associated with many neuropsychiatric disorders. Free phosphomonoesters and phosphodiesters, which can be detected by in vivo 31P magnetic resonance spectroscopy (MRS), are important cell membrane building blocks. However, the quantification of phosphoesters has been highly controversial even in healthy individuals due to overlapping signals from macromolecule membrane phospholipids (MP). In this study, high signal-to-noise ratio (SNR) cerebral 31P MRS spectra were acquired from healthy volunteers at both 3 and 7 Tesla. Our results indicated that, with minimal spectral interference from MP, the [phosphocreatine (PCr)]/[phosphocholine (PC) + glycerophosphocholine (GPC)] ratio measured at 7 Tesla agreed with its value expected from biochemical constraints. In contrast, the 3 Tesla [PCr]/[PC+GPC] ratio obtained using standard spectral fitting procedures was markedly smaller than the 7 Tesla ratio and than the expected value. The analysis suggests that the commonly used spectral model for MP may fail to capture its complex spectral features at 3 Tesla, and that additional prior knowledge is necessary to reliably quantify the phosphoester signals at low magnetic field strengths when spectral overlapping is significant.

Enhanced nuclear-spin hyperpolarization of amino acids and proteins via reductive radical quenchers.

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as an effective tool for the hyperpolarization of aromatic amino acids in solution, either in isolation or within proteins. One factor limiting the maximum achievable signal-to-noise ratio in LC-photo-CIDNP is the progressive degradation of the target molecule and photosensitizer upon long-term optical irradiation. Fortunately, this effect does not cause spectral distortions but leads to a progressively smaller signal buildup upon long-term data-collection (e.g. 500 nM tryptophan on a 600 MHz spectrometer after ca. 200 scans). Given that it is generally desirable to minimize the extent of photodamage, we report that low-µM amounts of the reductive radical quenchers vitamin C (VC, i.e., ascorbic acid) or 2-mercaptoethylamine (MEA) enable LC-photo-CIDNP data to be acquired for significantly longer time than ever possible before. This approach increases the sensitivity of LC-photo-CIDNP by more than 100%, with larger enhancement factors achieved in experiments involving more transients. Our results are consistent with VC and MEA acting primarily by reducing transient free radicals of the NMR molecule of interest, thus attenuating the extent of photodamage. The benefits of this reductive radical-quencher approach are highlighted by the ability to collect long-term high-resolution 2D 1H-13C LC-photo-CIDNP data on a dilute sample of the drkN SH3 protein (5 µM).

Backbone-independent NMR resonance assignments of methyl probes in large proteins.

Methyl-specific isotope labeling is a powerful tool to study the structure, dynamics and interactions of large proteins and protein complexes by solution-state NMR. However, widespread applications of this methodology have been limited by challenges in obtaining confident resonance assignments. Here, we present Methyl Assignments Using Satisfiability (MAUS), leveraging Nuclear Overhauser Effect cross-peak data, peak residue type classification and a known 3D structure or structural model to provide robust resonance assignments consistent with all the experimental inputs. Using data recorded for targets with known assignments in the 10-45 kDa size range, MAUS outperforms existing methods by up to 25,000 times in speed while maintaining 100% accuracy. We derive de novo assignments for multiple Cas9 nuclease domains, demonstrating that the methyl resonances of multi-domain proteins can be assigned accurately in a matter of days, while reducing biases introduced by manual pre-processing of the raw NOE data. MAUS is available through an online web-server.

In vivo absolute quantification of hepatic γ-ATP concentration in mice using 31 P MRS at 11.7 T.

Measurement of ATP concentrations and synthesis in humans indicated abnormal hepatic energy metabolism in obesity, non-alcoholic fatty liver disease (NAFLD) and Type 2 diabetes. Further mechanistic studies on energy metabolism require the detailed phenotyping of specific mouse models. Thus, this study aimed to establish and evaluate a robust and fast single voxel 31 P MRS method to quantify hepatic γ-ATP concentrations at 11.7 T in three mouse models with different insulin sensitivities and liver fat contents (72-week-old C57BL/6 control mice, 72-week-old insulin resistant sterol regulatory-element binding protein-1c overexpressing (SREBP-1c+ ) mice and 10-12-week-old prediabetic non-obese diabetic (NOD) mice). Absolute quantification was performed by employing an external reference and a matching replacement ATP phantom with 3D image selected in vivo spectroscopy 31 P MRS. This single voxel 31 P MRS method non-invasively quantified hepatic γ-ATP within 17 min and the repeatability tests provided a coefficient of variation of 7.8 ± 1.1%. The mean hepatic γ-ATP concentrations were markedly lower in SREBP-1c+ mice (1.14 ± 0.10 mM) than in C57BL/6 mice (2.15 ± 0.13 mM; p < 0.0002) and NOD mice (1.78 ± 0.13 mM; p < 0.006, one-way ANOVA test). In conclusion, this method allows us to rapidly and precisely measure hepatic γ-ATP concentrations, and thereby to non-invasively detect abnormal hepatic energy metabolism in mice with different degrees of insulin resistance and NAFLD. Thus, this 31 P MRS will also be useful for future mechanistic as well as therapeutic translational studies in other murine models.

Voxel-wise partial volume correction method for accurate estimation of tissue sodium concentration in 23 Na-MRI at 7 T.

Sodium is crucial for the maintenance of cell physiology, and its regulation of the sodium-potassium pump has implications for various neurological conditions. The distribution of sodium concentrations in tissue can be quantitatively evaluated by means of sodium MRI (23 Na-MRI). Despite its usefulness in diagnosing particular disease conditions, tissue sodium concentration (TSC) estimated from 23 Na-MRI can be strongly biased by partial volume effects (PVEs) that are induced by broad point spread functions (PSFs) as well as tissue fraction effects. In this work, we aimed to propose a robust voxel-wise partial volume correction (PVC) method for 23 Na-MRI. The method is based on a linear regression (LR) approach to correct for tissue fraction effects, but it utilizes a 3D kernel combined with a modified least trimmed square (3D-mLTS) method in order to minimize regression-induced inherent smoothing effects. We acquired 23 Na-MRI data with conventional Cartesian sampling at 7 T, and spill-over effects due to the PSF were considered prior to correcting for tissue fraction effects using 3D-mLTS. In the simulation, we found that the TSCs of gray matter (GM) and white matter (WM) were underestimated by 20% and 11% respectively without correcting tissue fraction effects, but the differences between ground truth and PVE-corrected data after the PVC using the 3D-mLTS method were only approximately 0.6% and 0.4% for GM and WM, respectively. The capability of the 3D-mLTS method was further demonstrated with in vivo 23 Na-MRI data, showing significantly lower regression errors (ie root mean squared error) as compared with conventional LR methods (p < 0.001). The results of simulation and in vivo experiments revealed that 3D-mLTS is superior for determining under- or overestimated TSCs while preserving anatomical details. This suggests that the 3D-mLTS method is well suited for the accurate determination of TSC, especially in small focal lesions associated with pathological conditions.

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies.

Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years.

1H detection of heteronuclear dipolar oscillations with water suppression in single crystal peptide and oriented protein samples.

Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples.

NMR Lineshape Analysis of Intrinsically Disordered Protein Interactions.

Interactions of intrinsically disordered proteins are central to their cellular functions, and solution-state NMR spectroscopy provides a powerful tool for characterizing both structural and mechanistic aspects of such interactions. Here we focus on the analysis of IDP interactions using NMR titration measurements. Changes in resonance lineshapes in two-dimensional NMR spectra upon titration with a ligand contain rich information on structural changes in the protein and the thermodynamics and kinetics of the interaction, as well as on the microscopic association mechanism. Here we present protocols for the optimal design of titration experiments, data acquisition, and data analysis by two-dimensional lineshape fitting using the TITAN software package.

Determining Binding Kinetics of Intrinsically Disordered Proteins by NMR Spectroscopy.

The unique structural flexibility of intrinsically disordered proteins (IDPs) is central to their diverse functions in cellular processes. Protein-protein interactions involving IDPs are frequently transient and dynamic in nature. Nuclear magnetic resonance (NMR) spectroscopy is an especially powerful tool for characterizing the structural propensities, dynamics, and interactions of IDPs. Here we describe applications of the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiment in combination with NMR titrations to characterize the kinetics and mechanisms of interactions between intrinsically disordered proteins and their targets. We illustrate the method with reference to interactions between the activation domain of the human T-cell leukemia virus type-I (HTLV-1) basic leucine zipper protein (HBZ) and its cellular binding partner, the KIX domain of the transcriptional coactivator CBP.

Multiple Site-Specific Phosphorylation of IDPs Monitored by NMR.

In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.

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.

In-Cell NMR of Intrinsically Disordered Proteins in Mammalian Cells.

In-cell NMR enables structural insights at atomic resolution of proteins in their natural environment. To date, very few methods have been developed to study proteins by in-cell NMR in mammalian systems. Here we describe a detailed protocol to conduct in-cell NMR on the intrinsically disordered protein of alpha-Synuclein (αSyn) in mammalian cells. This chapter includes a simplified expression and purification protocol of recombinant αSyn and its delivery into mammalian cells. The chapter also describes how to assess the cell leakage that might occur to the cells, the setup of the instrument, and how to perform basic analyses with the obtained NMR data.

Multi-receiver solid-state NMR using polarization optimized experiments (POE) at ultrafast magic angle spinning.

Ultrafast magic angle spinning (MAS) technology and 1H detection have dramatically enhanced the sensitivity of solid-state NMR (ssNMR) spectroscopy of biopolymers. We previously showed that, when combined with polarization optimized experiments (POE), these advancements enable the simultaneous acquisition of multi-dimensional 1H- or 13C-detected experiments using a single receiver. Here, we propose a new sub-class within the POE family, namely HC-DUMAS, HC-MEIOSIS, and HC-MAeSTOSO, that utilize dual receiver technology for the simultaneous detection of 1H and 13C nuclei. We also expand this approach to record 1H-, 13C-, and 15N-detected homonuclear 2D spectra simultaneously using three independent receivers. The combination of POE and multi-receiver technology will further shorten the total experimental time of ssNMR experiments for biological solids.

Effects of deuteration on solid-state NMR spectra of single peptide crystals and oriented protein samples.

Extensive deuteration can be used to simplify NMR spectra by "diluting" and minimizing the effects of the abundant 1H nuclei. In solution-state NMR and magic angle spinning solid-state NMR of proteins, perdeuteration has been widely applied and its effects are well understood. Oriented sample solid-state NMR of proteins, however, is at a much earlier stage of development. In spite of the promise of the approach, the effects of sample deuteration are largely unknown. Here we map out the effects of perdeuteration on solid-state NMR spectra of aligned samples by closely examining differences in results obtained on fully protiated and perdeuterated samples, where all of the carbon sites have either 1H or 2H bonded to them, respectively. The 2H and 15N labeled samples are back-exchanged in 1H2O solution so that the amide 15N sites have a bonded 1H. Line-widths in the 15N chemical shift, 1H chemical shift, and 1H-15N dipolar coupling frequency dimensions were compared for peptide single crystals as well as membrane proteins aligned along with the phospholipids in bilayers with their normals perpendicular to the direction of the magnetic field. Remarkably, line-width differences were not found between fully protiated and perdeuterated samples. However, in the absence of effective 1H-1H homonuclear decoupling, the line-widths in the 1H-15N heteronuclear dipolar coupling frequency dimension were greatly narrowed in the perdeuterated samples. In proton-driven spin diffusion (PDSD) experiments, no effects of perdeuteration were observed. In contrast, in mismatched Hartmann-Hahn experiments, perdeuteration enhances cross-peak intensities by allowing more efficient spin-exchange with less polarization transfer back to the carbon-bound 1H. Here we show that in oriented sample solid-state NMR, the effects of perdeuteration can be exploited in experiments where 1H-1H homonuclear decoupling cannot be applied. These data also provide evidence for the possible contribution of direct 15N-15N dilute-spin mixing mechanism in proton-driven spin diffusion experiments.

Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules.

In this review, lanthanide chelating tags and their applications to pseudocontact shift NMR spectroscopy as well as analysis of residual dipolar couplings are covered. A complete overview is presented of DOTA-derived and non-DOTA-derived lanthanide chelating tags, critical points in the design of lanthanide chelating tags as appropriate linker moieties, their stability under reductive conditions, e.g., for in-cell applications, the magnitude of the anisotropy transferred from the lanthanide chelating tag to the biomacromolecule under investigation and structural properties, as well as conformational bias of the lanthanide chelating tags are discussed. Furthermore, all DOTA-derived lanthanide chelating tags used for PCS NMR spectroscopy published to date are displayed in tabular form, including their anisotropy parameters, with all employed lanthanide ions, CB-Ln distances and tagging reaction conditions, i.e., the stoichiometry of lanthanide chelating tags, pH, buffer composition, temperature and reaction time. Additionally, applications of lanthanide chelating tags for pseudocontact shifts and residual dipolar couplings that have been reported for proteins, protein-protein and protein-ligand complexes, carbohydrates, carbohydrate-protein complexes, nucleic acids and nucleic acid-protein complexes are presented and critically reviewed. The vast and impressive range of applications of lanthanide chelating tags to structural investigations of biomacromolecules in solution clearly illustrates the significance of this particular field of research. The extension of the repertoire of lanthanide chelating tags from proteins to nucleic acids holds great promise for the determination of valuable structural parameters and further developments in characterizing intermolecular interactions.

Inductively coupled magic angle spinning microresonators benchmarked for high-resolution single embryo metabolomic profiling.

The magic angle coil spinning (MACS) technique has been introduced as a very promising extension for solid state NMR detection, demonstrating sensitivity enhancements by a factor of 14 from the very first time it has been reported. The main beneficiary of this technique is the scientific community dealing with mass- and volume-limited, rare, or expensive samples. However, more than a decade after the first report on MACS, there is a very limited number of groups who have continued to develop the technique, let alone it being widely adopted by practitioners. This might be due to several drawbacks associated with the MACS technology until now, including spectral linewidth, heating due to eddy currents, and imprecise manufacturing. Here, we report a device overcoming all these remaining issues, therefore achieving: (1) spectral resolution of approx 0.01 ppm and normalized limit of detection of approx. 13 nmol s0.5 calculated using the anomeric proton of sucrose at 3 kHz MAS frequency; (2) limited temperature increase inside the MACS insert of only 5 °C at 5 kHz MAS frequency in an 11.74 T magnetic field, rendering MACS suitable to study live biological samples. The wafer-scale fabrication process yields MACS inserts with reproducible properties, readily available to be used on a large scale in bio-chemistry labs. To illustrate the potential of these devices for metabolomic studies, we further report on: (3) ultra-fine 1H-1H and 13C-13C J-couplings resolved within 10 min for a 340 mM uniformly 13C-labeled glucose sample; and (4) single zebrafish embryo measurements through 1H-1H COSY within 4.5 h, opening the gate for the single embryo NMR studies.

Modular, triple-resonance, transmission line DNP MAS probe for 500 MHz/330 GHz.

We describe the design and construction of a modular, triple-resonance, fully balanced, DNP-MAS probe based on transmission line technology and its integration into a 500 MHz/330 GHz DNP-NMR spectrometer. A novel quantitative probe design and characterization strategy is developed and employed to achieve optimal sensitivity, RF homogeneity and excellent isolation between channels. The resulting three channel HCN probe has a modular design with each individual, swappable module being equipped with connectorized, transmission line ports. This strategy permits attachment of a mating connector that facilitates accurate impedance measurements at these ports and allows characterization and adjustment (e.g. for balancing or tuning/matching) of each component individually. The RF performance of the probe is excellent; for example, the 13C channel attains a Rabi frequency of 280 kHz for a 3.2 mm rotor. In addition, a frequency tunable 330 GHz gyrotron operating at the second harmonic of the electron cyclotron frequency was developed for DNP applications. Careful alignment of the corrugated waveguide led to minimal loss of the microwave power, and an enhancement factor ε = 180 was achieved for U-13C urea in the glassy matrix at 80 K. We demonstrated the operation of the system with acquisition of multidimensional spectra of cross-linked lysozyme crystals which are insoluble in glycerol-water mixtures used for DNP and samples of RNA.

Improved sensitivity and resolution of in-cell NMR spectra.

In-cell NMR spectroscopy is a powerful tool to study protein structures and interactions under near physiological conditions in both prokaryotic and eukaryotic living cells. The low sensitivity and resolution of in-cell NMR spectra and limited lifetime of cells over the course of an in-cell experiment have presented major hurdles to wide acceptance of the technique, limiting it to a few select systems. These issues are addressed by introducing the use of the CRINEPT pulse sequence to increase the sensitivity and resolution of in-cell NMR spectra and the use of a bioreactor to maintain cell viability for up to 24h. Application of advanced pulse sequences and bioreactor during in-cell NMR experiments will facilitate the exploration of a wide range of biological processes.

Aromatic 19F-13C TROSY: a background-free approach to probe biomolecular structure, function, and dynamics.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

Simple high-resolution NMR spectroscopy as a tool in molecular biology.

NMR is one of the major techniques for investigating the structure, dynamics and interactions between biomolecules. However, non-experts often experience NMR experimentation and data analysis as intimidating. We discuss a simple yet powerful NMR technique, the so-called chemical shift perturbation (CSP) analysis, as a tool to elucidate macromolecular interactions in small- and medium-sized complexes, including protein-protein, protein-drug, and protein-DNA/RNA interactions. We discuss current software packages for NMR data analysis and present a new interactive graphical tool implemented in CcpNmr AnalysisAssign version-3, which can drastically reduce the time required for the CSP analysis. Lastly, we illustrate the usefulness of a protein three-dimensional structure for interpretation of the CSP data.