RESUMO
The supramolecular interaction between lanthanide complexes and proteins is at the heart of numerous chemical and biological studies. Some of these complexes have demonstrated remarkable interaction properties with proteins or peptides in solution and in the crystalline state. Here we have used the paramagnetism of lanthanide ions to characterize the affinity of two lanthanide complexes for ubiquitin. As the interaction process is dynamic, the acquired NMR data only reflect the time average of the different steps. We have used molecular dynamics (MD) simulations to get a deeper insight into the detailed interaction scenario at the microsecond scale. This NMR/MD approach enabled us to establish that the tris-dipicolinate complex interacts specifically with arginines and lysines, while the crystallophore explores the protein surface through weak interactions with carboxylates. These observations shed new light on the dynamic interaction properties of these complexes, which will ultimately enable us to propose a crystallization mechanism.
Assuntos
Elementos da Série dos Lantanídeos , Simulação de Dinâmica Molecular , Ubiquitina , Ubiquitina/química , Elementos da Série dos Lantanídeos/química , Ressonância Magnética Nuclear Biomolecular , Ácidos Picolínicos/química , Ligação ProteicaRESUMO
Glutamine is under scrutiny regarding its metabolic deregulation linked to energetic reprogramming in cancer cells. Many analytical techniques have been used to better understand the impact of the metabolism of amino acids on biological processes, however only a few are suited to work with complex samples. Here, we report the use of a general dissolution dynamic nuclear polarization (D-DNP) formulation using an unexpensive radical as a multipurpose tool to study glutamine, with insights from enzymatic modelling to complex metabolic networks and fast imaging. First, hyperpolarized [5-13 C] glutamine is used as molecular probe to study the kinetic action of two enzymes: L-asparaginase that has been used as an anti-metabolic treatment for cancer, and glutaminase. These results are also compared with those acquired with another hyperpolarized amino acid, [1,4-13 C] asparagine. Second, we explored the use of hyperpolarized (HP) substrates to probe metabolic pathways by monitoring metabolic profiles arising from hyperpolarized glutamine in E.â coli extracts. Finally, a highly concentrated sample formulation is proposed for the purpose of fast imaging applications. We think that this approach can be extended to formulate other amino acids as well as other metabolites and provide complementary insights into the analysis of metabolic networks.
Assuntos
Escherichia coli , Glutamina , Glutamina/análise , Glutamina/química , Glutamina/metabolismo , Solubilidade , Escherichia coli/metabolismo , Redes e Vias Metabólicas , Aminoácidos/metabolismo , Isótopos de CarbonoRESUMO
Dynamical nuclear polarization (DNP) is a powerful method that allows one to polarize virtually any spin-bearing nucleus by transferring electron polarization by microwave irradiation of the electron Zeeman transitions. Under certain conditions, the DNP process can be described in thermodynamical terms using the thermal mixing (TM) model. Different nuclear species can exchange energy indirectly through their interactions with the electron spins and reach a common spin temperature. Such "cross-talk" effects can occur between proton (H) and deuterium (D) nuclei in de- and re-polarization experiments. In this work, we investigate such effects experimentally, using either protonated or deuterated TEMPOL radicals as polarizing agents. An analysis of these experiments based on Provotorov's equations allows one to extract the relevant kinetic parameters, such as the rates of energy transfer between the different reservoirs, and the heat capacity of the non-Zeeman (NZ) electron reservoir, while the heat capacities of the proton and deuterium reservoirs can be estimated based on their usual expressions. These parameters allow one to make predictions of the behaviour of heteronuclei such as carbon-13 or phosphorous-31, provided that their heat capacities are negligible. Finally, we present an experimental study of the dependence of Provotorov's kinetic parameters on the TEMPOL concentration and on the H/D ratio, thus providing insight into the nature of "hidden" spins that are not observable directly because of their proximity to the radicals.
RESUMO
Ultrahigh-resolution NMR has recently attracted considerable attention in the field of complex samples analysis. Indeed, the implementation of broadband homonuclear decoupling techniques has allowed us to greatly simplify crowded 1H spectra, yielding singlets for almost every proton site from the analyzed molecules. Pure shift methods have notably shown to be particularly suitable for deciphering mixtures of metabolites in biological samples. Here, we have successfully implemented a new pure shift pulse sequence based on the PSYCHE method, which incorporates a block for solvent suppression that is suitable for metabolomics analysis. The resulting experiment allows us to record ultrahigh-resolution 1D NOESY 1H spectra of biofluids with suppression of the water signal, which is a crucial step for highlighting metabolite mixtures in an aqueous phase. We have successfully recorded pure shift spectra on extracellular media of diffuse large B-cell lymphoma (DLBCL) cells. Despite a lower sensitivity, the resolution of pure shift data was found to be better than that of the standard approach, which provides a more detailed vision of the exo-metabolome. The statistical analyses carried out on the resulting metabolic profiles allow us to successfully highlight several metabolic pathways affected by these drugs. Notably, we show that Kidrolase plays a major role in the metabolic pathways of this DLBCL cell line.
Assuntos
Linfoma Difuso de Grandes Células B , Água , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Espectroscopia de Ressonância Magnética/métodos , Metaboloma , Metabolômica/métodosRESUMO
The 17O resonances of zirconium-oxo clusters that can be found in porous Zr carboxylate metal-organic frameworks (MOFs) have been investigated by magic-angle spinning (MAS) NMR spectroscopy enhanced by dynamic nuclear polarization (DNP). High-resolution 17O spectra at 0.037% natural abundance could be obtained in 48 hours, thanks to DNP enhancement of the 1H polarization by factors ε(1H) = Swith/Swithout = 28, followed by 1H â 17O cross-polarization, allowing a saving in experimental time by a factor of ca. 800. The distinct 17O sites from the oxo-clusters can be resolved at 18.8 T. Their assignment is supported by density functional theory (DFT) calculations of chemical shifts and quadrupolar parameters. Protonation of 17O sites seems to be leading to large characteristic shifts. Hence, natural abundance 17O NMR spectra of diamagnetic MOFs can thus be used to probe and characterize the local environment of different 17O sites on an atomic scale.
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Long-lived imbalances of spin state populations can circumvent fast quadrupolar relaxation by reducing effective longitudinal relaxation rates by about an order of magnitude. This opens new avenues for the study of dynamic processes in deuterated molecules. Here we present an analysis of the relaxation properties of deuterated methyl groups CD3. The number of coupled equations that describe cross-relaxation between the 27 symmetry-adapted spin states of a D3 system can be reduced to only 2 non-trivial "lumped modes" by (i) taking the sums of the populations of all states that equilibrate rapidly within each irreducible representation of the symmetry group, and (ii) by combining populations that have similar relaxation rates although they belong to different irreducible representations. The quadrupolar relaxation rates of the spin state imbalances in CD3 groups are determined not by the correlation time of overall tumbling of the molecule, but by the frequency of jumps of methyl groups about their three-fold symmetry axis. We access these states via dissolution dynamic nuclear polarization (D-DNP), a method that allows one to populate the desired long-lived states at cryogenic temperatures and their subsequent detection at ambient temperatures after rapid dissolution. Experimental examples of DMSO-d6 and ethanol-d6 demonstrate that long-lived deuterium spin states are indeed accessible and that their lifetimes can be determined. Our analysis of the system via "lumped" modes allows us to visualize different possible spin-state populations of symmetry A, B, or E. Thus, we identify a long-lived spin state involving all three deuterons in a CD3 group as an A/E imbalance that can be populated through DNP at low temperatures.
RESUMO
Tailoring the physical features and the porous network architecture of silica-based hyperpolarizing solids containing TEMPO radicals, known as HYPSO (hybrid polarizing solids), enabled unprecedented performance of dissolution dynamic nuclear polarization (d-DNP). High polarization values up to P(1 H)=99 % were reached for samples impregnated with a mixture of H2 O/D2 O and loaded in a 6.7â T polarizer at temperatures around 1.2â K. These HYPSO materials combine the best performance of homogeneous DNP formulations with the advantages of solid polarizing matrices, which provide hyperpolarized solutions free of any-potentially toxic-additives (radicals and glass-forming agents). The hyperpolarized solutions can be expelled from the porous solids, filtered, and rapidly transferred either to a nuclear magnetic resonance (NMR) spectrometer or to a magnetic resonance imaging (MRI) system.
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The columnar self-assembly resulting from units of N,N',N''-trihexylbenzene-1,3,5-tricarboxamide is investigated in solution and the solid state by means of NMR spectroscopy. A parallel computational study utilizing both semiempirical and DFT methods allows comparison between experimental results and calculated data for self-assembled and non-assembled structural hypotheses. The hybrid functional B3LYP is compared with the B3LYP-D and B97D functionals to assess contributions due to dispersion interactions. Interatomic distances are studied utilizing ROE experiments on proton spins in solution. Isotropic shifts as measured experimentally are shown to offer a method to assess the self-assemblies 'on-the-fly'. The anisotropic part of the shift interaction for carbon nuclei is probed in the solid state with specific magic-angle spinning experiments. The sensitivity of the NMR parameters for various carbon environments with respect to the orientation of the substituents and packing effects is investigated computationally. We show that all the utilized experimental techniques, in both solution and the solid state, and in combination with DFT calculations, are capable of discerning between assembled and non-assembled systems and offer a robust set of independent tools to highlight atomic details in self-organized structures.
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Dissolution dynamic nuclear polarization (D-DNP) has become a method of choice to enhance signals in nuclear magnetic resonance (NMR). Recently, we have proposed to combine cross-polarization (CP) with D-DNP to provide high polarization P(13C) in short build-up times. In this paper, we show that switching microwave irradiation off for a few hundreds of milliseconds prior to CP can significantly boost the efficiency. By implementing microwave gating, 13C polarizations on sodium [1-13C]acetate as high as 64% could be achieved with a polarization build-up time constant as short as 160 s. A polarization of P(13C) = 78% could even be reached for [13C]urea.
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Dipolar or quadrupolar echoes allow one to observe undistorted powder patterns, in contrast to simple Fourier transformations of free induction decays (FIDs). In this work, the buildup of proton polarization due to dynamic nuclear polarization (DNP) is monitored by observing echoes rather than FIDs. When the microwave irradiation is interrupted during the buildup of DNP, the electrons relax back to their Boltzmann distribution at high fields (B0 = 6.7 T) and low temperatures 1.2 < Tsample < 4.0 K, so that dipolar flip-flop-flip terms involving two electrons and one proton become largely ineffective as a mechanism of proton decoherence. This leads to a prolongation of the nuclear coherence lifetime T2'(1H). The increase in T2'(1H) leads to transient surges of the amplitudes of spin echoes. Conversely, transient slumps of spin echoes are observed when the microwave irradiation is switched back on, due to a shortening of nuclear coherence lifetimes.
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Dynamic nuclear polarization of samples at low temperatures, typically between 1.2 and 4.2 K, allows one to achieve spin temperatures of as low as 2 mK so that for many nuclear isotopes the high-temperature approximation is violated for the nuclear Zeeman interaction. This leads to characteristic asymmetries in powder spectra. We show that the line shapes due to the quadrupolar couplings of deuterium spins present in virtually all solvents used for such experiments (DNP juice) allow the quick yet accurate determination of the deuterium spin temperature or, equivalently, the deuterium polarization. The observation of quadrupolar echoes excited by small flip-angle pulses allows one to monitor the build-up and decay of the positive or negative deuterium polarization.
RESUMO
A novel system for dissolution dynamic nuclear polarization based on a cost-effective "cryogen-free" magnet that can generate fields up to 9.4â¯T with a sample space that can reach temperatures below 1.4â¯K in a continuous and stable manner. Polarization levels up to P(1H)â¯=â¯60⯱â¯5% can be reached with TEMPOL in about 20â¯min, and P(13C)â¯=â¯50⯱â¯5% can be achieved using adiabatic cross polarization.