Reversible Parahydrogen Induced Hyperpolarization of 15 N in Unmodified Amino Acids Unraveled at High Magnetic Field.

Amino acids (AAs) and ammonia are metabolic markers essential for nitrogen metabolism and cell regulation in both plants and humans. NMR provides interesting opportunities to investigate these metabolic pathways, yet lacks sensitivity, especially in case of 15 N. In this study, spin order embedded in p-H2 is used to produce on-demand reversible hyperpolarization in 15 N of pristine alanine and ammonia under ambient protic conditions directly in the NMR spectrometer. This is made possible by designing a mixed-ligand Ir-catalyst, selectively ligating the amino group of AA by exploiting ammonia as a strongly competitive co-ligand and preventing deactivation of Ir by bidentate ligation of AA. The stereoisomerism of the catalyst complexes is determined by hydride fingerprinting using 1 H/D scrambling of the associated N-functional groups on the catalyst (i.e., isotopological fingerprinting), and unravelled by 2D-ZQ-NMR. Monitoring the transfer of spin order from p-H2 to 15 N nuclei of ligated and free alanine and ammonia targets using SABRE-INEPT with variable exchange delays pinpoints the monodentate elucidated catalyst complexes to be most SABRE active. Also RF-spin locking (SABRE-SLIC) enables transfer of hyperpolarization to 15 N. The presented high-field approach can be a valuable alternative to SABRE-SHEATH techniques since the obtained catalytic insights (stereochemistry and kinetics) will remain valid at ultra-low magnetic fields.

Convergent views on disordered protein dynamics from NMR and computational approaches.

Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.

Isotopological Fingerprinting Using 1H/D Scrambling Identifies the Stereochemistry of Hyperpolarization Catalysts Transferring Spin Polarization from Parahydrogen to Substrates Using Signal Amplification by Reversible Exchange.

Hyperpolarization using signal amplification by reversible exchange (SABRE) relies on target molecules and parahydrogen coordinating to a transition metal catalyst. Identification of this coordinated state becomes increasingly important, especially since bio-relevant targets such as pyruvate and amino acids exhibiting multiple binding sites are becoming compatible with SABRE. In this report, we present a fingerprinting method to discriminate and identify ligand binding sites without requiring the presence of a sensitive or isotope-labeled heteroatom. Adding a small concentration of protons to a deuterated medium, spontaneous 1H/D scrambling of exchangeable protons encodes the ligands each with an isotopological fingerprint. By use of rapid 2D zero quantum NMR, the binding sites are decoded from the hydrides in less than a minute. The new methodology is explained and demonstrated on Ir mixed complexes with pyridine, benzylamine, and ammonia as common N-functional ligands.

Long-Term Generation of Longitudinal Spin Order Controlled by Ammonia Ligation Enables Rapid SABRE Hyperpolarized 2D NMR.

The front cover artwork front cover artwork is provided by NMRCoRe, the Flemish NMR/X-Ray platform for Convergence Research and was designed by Ir. Ewoud Vaneeckhaute and Dr. Eric Breynaert. The image shows the reciprocity between parahydrogen, deuterated ammonia and iridium allowing for hyperpolarized 2D NMR via long-term availability of longitudinal spin order. Read the full text of the Article at 10.1002/cphc.202100079.

Detection of Metabolite-Protein Interactions in Complex Biological Samples by High-Resolution Relaxometry: Toward Interactomics by NMR.

Metabolomics, the systematic investigation of metabolites in biological fluids, cells, or tissues, reveals essential information about metabolism and diseases. Metabolites have functional roles in a myriad of biological processes, as substrates and products of enzymatic reactions but also as cofactors and regulators of large numbers of biochemical mechanisms. These functions involve interactions of metabolites with macromolecules. Yet, methods to systematically investigate these interactions are still scarce to date. In particular, there is a need for techniques suited to identify and characterize weak metabolite-macromolecule interactions directly in complex media such as biological fluids. Here, we introduce a method to investigate weak interactions between metabolites and macromolecules in biological fluids. Our approach is based on high-resolution NMR relaxometry and does not require any invasive procedure or separation step. We show that we can detect interactions between small and large molecules in human blood serum and quantify the size of the complex. Our work opens the way for investigations of metabolite (or other small molecules)-protein interactions in biological fluids for interactomics or pharmaceutical applications.

Long-Term Generation of Longitudinal Spin Order Controlled by Ammonia Ligation Enables Rapid SABRE Hyperpolarized 2D NMR.

Symmetry breaking of parahydrogen using iridium catalysts converts singlet spin order into observable hyperpolarization. In this contribution, iridium catalysts are designed to exhibit asymmetry in their hydrides, regulated by in situ generation of deuterated ammonia governed by ammonium buffers. The concentrations of ammonia (N) and pyridine (P) provide a handle to generate a variety of stereo-chemically asymmetric N-heterocyclic carbene iridium complexes, ligating either [3xP], [2xP;N], [P;2xN] or [3xN] in an octahedral SABRE type configuration. The non-equivalent hydride positions, in correspondence with the ammonium buffer solutions, enables to extend singlet-triplet or S⟩→T0⟩ mixing at high magnetic field and experimentally induce prolonged generation of non-equilibrium longitudinal two-spin order. This long-lasting magnetization can be exploited in hyperpolarized 2D-OPSY-COSY experiments providing direct structural information on the catalyst using a single contact with parahydrogen. Separately, field cycling revealed hyperpolarization properties in low-field conditions. Controlling catalyst stereochemistry by introducing small and deuterated ligands, such as deuterated ammonia, simplifies the spin-system. This is shown to unify experimental and theoretically derived field-sweep experiments for four-spin systems.

Boosting the resolution of low-field [Formula: see text] relaxation experiments on intrinsically disordered proteins with triple-resonance NMR.

Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone [Formula: see text] amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of [Formula: see text] subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.

Protein Dynamics from Accurate Low-Field Site-Specific Longitudinal and Transverse Nuclear Spin Relaxation.

Nuclear magnetic relaxation provides invaluable quantitative site-specific information on the dynamics of complex systems. Determining dynamics on nanosecond time scales requires relaxation measurements at low magnetic fields incompatible with high-resolution NMR. Here, we use a two-field NMR spectrometer to measure carbon-13 transverse and longitudinal relaxation rates at a field as low as 0.33 T (proton Larmor frequency 14 MHz) in specifically labeled side chains of the protein ubiquitin. The use of radiofrequency pulses enhances the accuracy of measurements as compared to high-resolution relaxometry approaches, where the sample is moved in the stray field of the superconducting magnet. Importantly, we demonstrate that accurate measurements at a single low magnetic field provide enough information to characterize complex motions on low nanosecond time scales, which opens a new window for the determination of site-specific nanosecond motions in complex systems such as proteins.

Time-Resolved Protein Side-Chain Motions Unraveled by High-Resolution Relaxometry and Molecular Dynamics Simulations.

Motions of proteins are essential for the performance of their functions. Aliphatic protein side chains and their motions play critical roles in protein interactions: for recognition and binding of partner molecules at the surface or serving as an entropy reservoir within the hydrophobic core. Here, we present a new NMR method based on high-resolution relaxometry and high-field relaxation to determine quantitatively both motional amplitudes and time scales of methyl-bearing side chains in the picosecond-to-nanosecond range. We detect a wide variety of motions in isoleucine side chains in the protein ubiquitin. We unambiguously identify slow motions in the low nanosecond range, which, in conjunction with molecular dynamics computer simulations, could be assigned to transitions between rotamers. Our approach provides unmatched detailed insight into the motions of aliphatic side chains in proteins and provides a better understanding of the nature and functional role of protein side-chain motions.

DOSY Analysis of Micromolar Analytes: Resolving Dilute Mixtures by SABRE Hyperpolarization.

DOSY is an NMR spectroscopy technique that resolves resonances according to the analytes' diffusion coefficients. It has found use in correlating NMR signals and estimating the number of components in mixtures. Applications of DOSY in dilute mixtures are, however, held back by excessively long measurement times. We demonstrate herein, how the enhanced NMR sensitivity provided by SABRE hyperpolarization allows DOSY analysis of low-micromolar mixtures, thus reducing the concentration requirements by at least 100-fold.

Full Correlations across Broad NMR Spectra by Two-Field Total Correlation Spectroscopy.

Total correlation spectroscopy (TOCSY) is a key experiment to assign nuclear magnetic resonance (NMR) spectra of complex molecules. Carbon-13 TOCSY experiments are essential to assign signals of protein side chains. However, the performance of carbon-13 TOCSY deteriorates at high magnetic fields since the necessarily limited radiofrequency irradiation fails to cover the broad range of carbon-13 frequencies. Here, we introduce a new concept to overcome the limitations of TOCSY by using two-field NMR spectroscopy. In two-field TOCSY experiments, chemical shifts are labelled at high field but isotropic mixing is performed at a much lower magnetic field, where the frequency range of the spectrum is drastically reduced. We obtain complete correlations between all carbon-13 nuclei belonging to amino acids across the entire spectrum: aromatic, aliphatic and carboxylic. Two-field TOCSY should be a robust and general approach for the assignment of uniformly carbon-13 labelled molecules in high-field and ultra-high field NMR spectrometers beyond 1000 MHz.

High-resolution two-field nuclear magnetic resonance spectroscopy.

Nuclear magnetic resonance (NMR) is a ubiquitous branch of spectroscopy that can explore matter at the scale of an atom. Significant improvements in sensitivity and resolution have been driven by a steady increase of static magnetic field strengths. However, some properties of nuclei may be more favourable at low magnetic fields. For example, transverse relaxation due to chemical shift anisotropy increases sharply at higher magnetic fields leading to line-broadening and inefficient coherence transfers. Here, we present a two-field NMR spectrometer that permits the application of rf-pulses and acquisition of NMR signals in two magnetic centres. Our prototype operates at 14.1 T and 0.33 T. The main features of this system are demonstrated by novel NMR experiments, in particular a proof-of-concept correlation between zero-quantum coherences at low magnetic field and single quantum coherences at high magnetic field, so that high resolution can be achieved in both dimensions, despite a ca. 10 ppm inhomogeneity of the low-field centre. Two-field NMR spectroscopy offers the possibility to circumvent the limits of high magnetic fields, while benefiting from their exceptional sensitivity and resolution. This approach opens new avenues for NMR above 1 GHz.

Recovering Invisible Signals by Two-Field NMR Spectroscopy.

Nuclear magnetic resonance (NMR) studies have benefited tremendously from the steady increase in the strength of magnetic fields. Spectacular improvements in both sensitivity and resolution have enabled the investigation of molecular systems of rising complexity. At very high fields, this progress may be jeopardized by line broadening, which is due to chemical exchange or relaxation by chemical shift anisotropy. In this work, we introduce a two-field NMR spectrometer designed for both excitation and observation of nuclear spins in two distinct magnetic fields in a single experiment. NMR spectra of several small molecules as well as a protein were obtained, with two dimensions acquired at vastly different magnetic fields. Resonances of exchanging groups that are broadened beyond recognition at high field can be sharpened to narrow peaks in the low-field dimension. Two-field NMR spectroscopy enables the measurement of chemical shifts at optimal fields and the study of molecular systems that suffer from internal dynamics, and opens new avenues for NMR spectroscopy at very high magnetic fields.

Optimization of SABRE for polarization of the tuberculosis drugs pyrazinamide and isoniazid.

Hyperpolarization produces nuclear spin polarization that is several orders of magnitude larger than that achieved at thermal equilibrium thus providing extraordinary contrast and sensitivity. As a parahydrogen induced polarization (PHIP) technique that does not require chemical modification of the substrate to polarize, Signal Amplification by Reversible Exchange (SABRE) has attracted a lot of attention. Using a prototype parahydrogen polarizer, we polarize two drugs used in the treatment of tuberculosis, namely pyrazinamide and isoniazid. We examine this approach in four solvents, methanol-d4, methanol, ethanol and DMSO and optimize the polarization transfer magnetic field strength, the temperature as well as intensity and duration of hydrogen bubbling to achieve the best overall signal enhancement and hence hyperpolarization level.