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. In solution state, as the molecular weight of analytes increases, coherences with long lifetimes are needed to yield advantageous through-space magnetisation transfers. Interactions between magnetic nuclei can only be detected provided the lifetimes of spin order are sufficient. In J-coupled pairs of nuclei, long-lived coherences (LLC's) connect states with different spin-permutation symmetry. Here in, we show sustained LLC's in protein Lysozyme, weighing 14.3 kDa, with lifetimes twice as long as those of classical magnetisation for the aliphatic protons of glycine residues. We found for the first time that, in a protein of significant molecular weight, LLC's yield substantial through-space magnetisation transfers: spin-order transfer stemming from LLC's overcame transfers from classical coherences by factors > 2. Furthermore, in agreement with theory, the permutation symmetry of LLC-based transfers allows mapping interacting atoms in the protein structure with respect to the molecular plane of glycine residues in a stereospecific manner. These findings can extend the scope of liquid-state high-resolution biomolecular spectroscopy.

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. We demonstrate this experiment for the follow-up of glutathione oxidation kinetics. On-the-fly stroboscopic detection minimizes the durations necessary for single acquisitions yet necessitates sustaining of magnetization lifetimes. Long-lived proton spin states (LLS) in the cysteine and glycine residues of glutathione with TLLS up to 16 s are reached. Based on 1H LLS, we followed fast oxidation kinetics in the glutathione redox pair GSH/GSSG. This new detection method allows sampling of long-lived spin order multiple times via small flip-angle excitations. This establishes the ground for the follow-up of redox processes detecting GSH/GSSG kinetics as magnetic-resonance biomarker of FLASH oxidative processes on time scales of tens of seconds.

Selective Excitation of Long-Lived Nuclear Spin States.

Nuclear magnetization storage, once limited by longitudinal and transverse relaxation lifetimes, T1 and T2, 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 T1 and T2, respectively. Excitation and/or detection of LLS currently involves pulses covering wide frequency ranges in high-magnetic-field spectrometers. This leads to excitation of unwanted signals that may overlap and interfere with the resonances of interest. Herein, we present a new pulse sequence that converts longitudinal magnetization to LLS and further to detectable magnetization using only frequency-selective pulses. We demonstrate the suitability of this sequence for different J-coupled spin pairs in dipeptide AlaGly and protein Ubiquitin. The newly developed method is adapted for investigations of LLS in complex systems such as proteins and mixtures of metabolites where selected molecular groups are to be investigated separately.

Offsets between hyperconjugations, p→d donations and Pauli repulsions impact the bonding of E-O-E systems. Case study on elements of Group 14.

The nature of the E-O chemical bond (E = C, Si, Ge, Sn) is investigated in a wide range of model derivatives, such as oxonium cations, hydrogenated/methylated/fluorinated/chlorinated ethers and acyclic oligomers incorporating the E-O-E moiety. By means of density functional theory (DFT) calculations and natural bond orbital (NBO) techniques, we propose a bonding mechanism that explains the structural contrast between the organic and the inorganic counterparts of all these derivatives: the interplay between stabilizing interactions like LP(O)→σ*(E-X) hyperconjugations and LP(O)→d(E) donations with LP(O)⋯σ(E-X) vicinal Pauli repulsions (X = H, C, O, F, Cl) dictates the equilibrium structures in terms of E-O-E angles and E-O bond lengths. In addition, the present work represents the first study of oxonium ions that describes the structural discrepancies among organic derivatives and their heavier analogues. Another novel outcome for ethers and oligomers is that the two non-equivalent lone pair electrons (LPs) at the oxygen atoms impact in different manners the geometries of such derivatives, i.e. the s/p LP is correlated with the bending behaviour of the E-O-E units, while the pure p LP mainly dictates the short E-O bond distances of inorganic derivatives. Lastly, we evaluate the impact of the number of electronegative substituents, e.g. F, Cl or OEH3 groups, on the bond patterns developed for hydrogenated or methylated ethers.

Symmetry versus entropy: Long-lived states and coherences.

In recent years, new molecular symmetry-based approaches for magnetic resonance have been invented. The implications of these discoveries will be significant for molecular imaging via magnetic resonance, in vitro as well as in vivo, for quantum computing and for other fields. Since the initial observation in 2004 in Southampton that effective spin symmetry can be instilled in a molecule during magnetic resonance experiments, spin states that are resilient to relaxation mechanisms have been increasingly used. Most of these states are related to the nuclear singlet in a pair of J-coupled spins. Tailored relaxation rate constants for magnetization became available in molecules of different sizes and structures, as experimental developments broadened the scope of symmetry-adapted spin states. The ensuing access to timescales longer than the classically-attained ones by circa one order of magnitude allows the study of processes such as slow diffusion or slow exchange that were previously beyond reach. Long-lived states formed by differences between populations of singlets and triplets have overcome the limitations imposed by longitudinal relaxation times (T1) by factors up to 40. Long-lived coherences formed by superpositions of singlets and triplets have overcome the limit of classical transverse coherence (T2) by a factor 9. We present here an overview of the development and applications of long-lived states (LLS) and long-lived coherences (LLC's) and considerations on future perspectives.

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. We propose to reveal herein the part of discoveries that may have been obscured by our choice to only look at them through the experience of such pre-established directions at the time. The methodological developments that are emphasised herein are the mechanisms of translation between the symmetric and non-symmetric environments with respect to the main magnetic field B0. More specifically, we look again thoroughly at zero-quantum rotations in the starting blocks of long-lived state populations, magnetisation transfers between hyperpolarised heteronuclei, and protons. These pulse sequences seed subsequent magnetic mechanisms that contribute to further applications. For instance, we show how some of the introduced coherence rotations were combined with classical pulse blocks to obtain two-dimensional correlations between protons and heteronuclei. We hope the pulse sequence building blocks discussed herein will open further perspectives for magnetic resonance experiments with long-lived spin order.

Theoretical Insights into the Structural Differences between Organic and Inorganic Amines/Ethers.

The contrasting geometrical features between organic and inorganic counterparts of amines and oxanes are explained in terms of an offset between attractive (donor-acceptor) and repulsive (donor-donor) interactions. Natural bond orbital (NBO) calculations carried out at the density functional theory level of theory reveal that hyperconjugative effects in the organic amines and ethers are overcome by repulsive interactions occurring between the lone pair on the nitrogen/oxygen atom and the adjacent σ(C-R) bond orbitals. Although displaying lower energies than in the corresponding organic derivatives, the LP(X) → σ*(E-R) (X = N, O; E = Si, Ge, Sn) interactions in heavier counterparts overcome the LP(X)···σ(E-R) repulsions, impacting thus their structural behavior. In addition, NBO deletion optimizations emphasize that among hyperconjugations, back-bonding effects of the LP(X) → d(E) type dictate to a lesser extent the anomalous structures of the inorganic amines and oxanes.

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. The new spectroscopic method we report allows to locate water-originating hydrogens in peptides and proteins via their effect on nuclear magnetic transitions similar to electronic phosphorescence, long-lived coherences. The sign of long-lived coherences excited in coupled protons can be switched by the experimenter. The different effect of water-exchanging hydrogens on long-lived coherences with opposed signs allows to pinpoint the position of these labile hydrogen atoms in the molecular framework of peptides and proteins.