Improved detection of magnetic interactions in proteins based on long-lived coherences.

Living systems rely on molecular building blocks with low structural symmetry. Therefore, constituent amino acids and nucleotides yield short-lived nuclear magnetic responses to electromagnetic radiation. Magnetic signals are at the basis of molecular imaging, structure determination and interaction studies.

Earth's field NMR relaxation of pre-polarised water protons for real-time detection of free-radical formation.

Real-time imaging of free-radical formation is important in physical chemistry, biochemistry, and radiobiology, especially for the study of radiation dose-rate effects. Herein, we show for the first time that the formation of free radicals during the time course of a chemical reaction can be imaged through NMR relaxation measurements of water protons in the Earth's magnetic field, in an open-coil spectrometer. The relaxation rate constants of water magnetisation are enhanced as reactions leading to the formation of hydroxyl radicals and oxygen proceed on the timescale of tens of minutes.

Multiple Stroboscopic Detection of Long-Lived Nuclear Magnetization for Glutathione Oxidation Kinetics.

Imaging the molecular kinetics of antioxidants by magnetic resonance can contribute to the mechanistic understanding of therapeutic approaches. Magnetic resonance detection of the response to flashes of oxidative stress requires sequential spectroscopy on the same time scale on which reactive oxygen species are generated. To this effect, we propose a single-polarization multiple-detection stroboscopic experiment.

Selective Excitation of Long-Lived Nuclear Spin States.

Nuclear magnetization storage, once limited by longitudinal and transverse relaxation lifetimes, and , can be prolonged by symmetry-adapted nuclear spin order, i.e. long-lived states (LLS) and long-lived coherences (LLC), which have significantly extended relaxation time constants compared to and , respectively.

Mechanisms of coherent re-arrangement for long-lived spin order.

Long-lived spin order-based approaches for magnetic resonance rely on the transition between two magnetic environments of different symmetries, one governed by the magnetic field of the spectrometer and the other where this strong magnetic field is inconsequential. Research on the excitation of magnetic-symmetry transitions in nuclear spins is a scientific field that debuted in Southampton in the year 2000. We advanced in this field carrying the baggage of pre-established directions in NMR spectroscopy.

Water hydrogen uptake in biomolecules detected via nuclear magnetic phosphorescence.

We introduce a new symmetry-based method for structural investigations of areas surrounding water-exchanging hydrogens in biomolecules by liquid-state nuclear magnetic resonance spectroscopy. Native structures of peptides and proteins can be solved by NMR with fair resolution, with the notable exception of labile hydrogen sites. The reason why biomolecular structures often remain elusive around exchangeable protons is that the dynamics of their exchange with the solvent hampers the observation of their signals.

Hyperpolarized Water Enhances Two-Dimensional Proton NMR Correlations: A New Approach for Molecular Interactions.

Protein and peptide interactions are characterized in the liquid state by multidimensional NMR spectroscopy experiments, which can take hours to record. We show that starting from hyperpolarized HDO, two-dimensional (2D) proton correlation maps of a peptide, either free in solution or interacting with liposomes, can be acquired in less than 60 s. In standard 2D NMR spectroscopy without hyperpolarization, the acquisition time required for similar spectral correlations is on the order of hours.

Laser-driven radiation: Biomarkers for molecular imaging of high dose-rate effects.

Recently developed short-pulsed laser sources garner high dose-rate beams such as energetic ions and electrons, x rays, and gamma rays. The biological effects of laser-generated ion beams observed in recent studies are different from those triggered by radiation generated using classical accelerators or sources, and this difference can be used to develop new strategies for cancer radiotherapy. High-power lasers can now deliver particles in doses of up to several Gy within nanoseconds.

Hyperpolarised NMR to follow water proton transport through membrane channels via exchange with biomolecules.

Water uptake in vesicles and the subsequent exchange between water protons and amide -NH protons in amino acids can be followed by a new, highly sensitive, type of magnetic resonance spectroscopy: dynamic nuclear polarisation (DNP)-enhanced NMR in the liquid state. Water hydrogen atoms are detected prior to and after their transfer to molecular sites in peptides and proteins featuring highly-accessible proton-exchangeable groups, as is the case for the -NH groups of intrinsically disordered proteins. The detected rates for amide proton-water proton exchange can be modulated by membrane-crossing rates, when a membrane channel is interposed.

Long-lived states detect interactions between small molecules and diamagnetic metal ions.

Long-lived states of nuclear spin order were used for the first time to probe interactions between molecules and diamagnetic metal ions. Proton spin states with lifetimes twice as long as the spin-lattice relaxation time constants of the same nuclei were promoted on the methoxyphenyl and tolyl substituents of a 1,3,4-oxadiazole derivative. The transient interaction of this oxadiazole derivative with silver(I) ions significantly speeds up the relaxation rate constants of proton long-lived states.

Pyruvate cellular uptake and enzymatic conversion probed by dissolution DNP-NMR: the impact of overexpressed membrane transporters.

Pyruvate membrane crossing and its lactate dehydrogenase-mediated conversion to lactate in cells featuring different levels of expression of membrane monocarboxylate transporters (MCT4) were probed by dissolution dynamic nuclear polarization-enhanced NMR. Hyperpolarized C-1-labeled pyruvate was transferred to suspensions of rodent tumor cell carcinoma, cell line 39. The pyruvate-to-lactate conversion rate monitored by dissolution dynamic nuclear polarization-NMR in carcinoma cells featuring native MCT4 expression level was lower than the rate observed for cells in which the human MCT4 gene was overexpressed.

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer.

Extending timescales and narrowing linewidths in NMR.

Among the different fields of research in nuclear magnetic resonance (NMR) which are currently investigated in the Laboratory of Biomolecular Magnetic Resonance (LRMB), two subjects that are closely related to each other are presented in this article. On the one hand, we show how to populate long-lived states (LLS) that have long lifetimes T(LLS) which allow one to go beyond the usual limits imposed by the longitudinal relaxation time T1. This makes it possible to extend NMR experiments to longer time-scales.

Long-lived states to monitor protein unfolding by proton NMR.

The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state.

Proton hyperpolarisation preserved in long-lived states.

The polarisation of abundant protons, rather than dilute nuclei with low gyromagnetic ratios, can be enhanced in less than 10 min using dissolution DNP and converted into a long-lived state delocalised over an ensemble of three coupled protons. The process is more straightforward than the hyperpolarisation of heteronuclei followed by magnetisation transfer to protons.

Long-lived coherences for homogeneous line narrowing in spectroscopy.

Line broadening, which can arise from inhomogeneities or homogeneous relaxation effects that lead to finite lifetimes of quantum states, is the Achilles' heel of many forms of spectroscopy. We show that line broadening may be considerably reduced by exploiting long lifetimes associated with superpositions of quantum states with different symmetry, termed long-lived coherences. In proton NMR of arbitrary molecules (including proteins) in isotropic solution, the slow oscillatory decays of long-lived coherences can yield spectra with very high resolution.

Proton NMR of (15)N-choline metabolites enhanced by dynamic nuclear polarization.

Chemical shifts of protons can report on metabolic transformations such as the conversion of choline to phosphocholine. To follow such processes in vivo, magnetization can be enhanced by dynamic nuclear polarization (DNP). We have hyperpolarized in this manner nitrogen-15 spins in (15)N-labeled choline up to 3.

Diffusion coefficients of biomolecules using long-lived spin states.

We report the first observation of long-lived states (LLS) having lifetimes T(LLS) that exceed the corresponding spin-lattice relaxation times T(1) by more than a factor 6 in a protein. Slow diffusion coefficients characteristic of large biomolecules can be determined by combining LLS methods with moderate pulsed field gradients (PFGs) available on commercial probeheads, as the extension of spin memory reduces the strain on the duration and/or strength of the PFGs. No isotope labeling of the biomolecule is necessary.

Measurement of slow diffusion coefficients of molecules with arbitrary scalar couplings via long-lived spin states.

New experiments are described for the determination of very slow diffusion constants by nuclear magnetic resonance (NMR) using long-lived (singlet) states. These experiments are suitable for molecules or conformations featuring a wide range of J-couplings.

Single or triple gradients?

Pulsed Field Gradients (PFGs) have become ubiquitous tools not only for Magnetic Resonance Imaging (MRI), but also for NMR experiments designed to study translational diffusion, for spatial encoding in ultra-fast spectroscopy, for the selection of desirable coherence transfer pathways, for the suppression of solvent signals, and for the elimination of zero-quantum coherences. Some of these experiments can only be carried out if three orthogonal gradients are available, while others can also be implemented using a single gradient, albeit at some expense of performance. This paper discusses some of the advantages of triple- with respect to single-gradient probes.