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Spin-alignment alternatives: Nuclear Hyperpolarization by para Hydrogen & optical pumping
Apart from polarization transfers from unpaired electrons reservoir to achieve nuclear hyperpolarization, another source of polarization is the H2 molecule itself. Hydrogen molecule exists at room temperature as two isomeric forms: ortho and para-hydrogen. Although para-hydrogen itself has no net spin angular momentum and is NMR silent, it can be used as a reagent to create reaction products that possess non- Boltzmann nuclear distributions with high degrees of spin alignment. Hydrogen can be enriched in the para form at low temperature in the presence of a paramagnetic catalyst.
Following the addition of para-H2 to an unsaturated substrate, the perturbation of the spin populations is maintained in the product, and typical NMR spectra characterized by strongly enhanced signals and adsorption-emission patterns are observed. This procedure is termed as para-Hydrogen-induced polarization (PHIP) and is limited to molecules with unsaturation double or triple bonds [114, 115]. Depending on whether the chemical reaction is conducted in the high or very low magnetic field there are two protocols leading to different signal patterns, named PASADENA (Parahydrogen And Synthesis Allow Dramatically Enhanced Nuclear Alignment) and ALTADENA (Adiabatic Longitudinal Transfer After Dissociation Engenders Nuclear Alignment). Another alternative is to transfer the para-H2 derived spin order to other molecules via reversible interactions, without hydrogenation termed as SABRE (Signal Amplification By Reversible Exchange) [116].
An important current trend in solid-state nuclear magnetic resonance is also the exploitation of optical pumping of nuclear spin polarizations as a means of enhancing and localizing NMR signals. Recent work in this domain focuses mainly in two areas, namely optically pumped NMR in semiconductors and optical pumping of noble gases. Xenon (129Xe) is an attractive substrate because it is usually not present inliving organisms and optical pumping is a rather easy method for imparting hyperpolarization, and because it can resonate over a wide range of chemical shifts, thus being an accurate reporter for changes in its proximity. Introducing optically pumped noble gases can boost NMR sensitivity by three to four orders of magnitude.
In addition 129Xe is particularly well suited for biosensing applications because it is a nontoxic element that retains its laser-induced polarization for relatively long time periods [117].
Heteronuclear Overhauser Effect in organic solids
The use of nuclear Overhauser effect (NOE) [132, 133] as a tool for structural elucidation has been extensively demonstrated in solution-state NMR [134]. It is one of the most widely exploited phenomena in solution-state NMR but NOEs in the solid state are rare. The effect arises through cross-relaxation driven by the modulation of the dipolar interaction between two spatially proximate spins, conventionally labelled I and S. Upon perturbation of the populations of the I-spin energy levels, crossrelaxation processes act to return the populations of the energy levels to their equilibrium values. This has the simultaneous effect of altering the population differences across the S-spin system through zero- and double quantum transitions, so leading to the observed change in S-spin signal intensity. This says that for zero mixing time (τm) the S magnetization is equal to its equilibrium value, but that as the mixing time increases the S magnetization has an additional contribution depending on the mixing time and the cross-relaxation rate, σIS. This latter term results in a change in the intensity of the S spin signal, and this change is called an NOE enhancement.
The normal procedure for visualizing these enhancements is to record first a reference spectrum in which the intensities are unperturbed. Modulation of the dipole-dipole interaction arises due to the presence of molecular motion on the appropriate timescale. In the solution state, this motion exists as a result of the rapid, random tumbling. In the solid state, such motions are rarely observed due to the structural rigidity imposed on the molecules, so mainly the rapid rotation of methyl groups or modulation of dipolar interactions by rapid molecular tumbling leads to enhancement of the 13C/15N signal intensity.
Revealing the sources of the heteronuclear Overhauser enhancement
To confirm the presence of heteronuclear Overhauser effects and identify the sources of the intensity enhancement, while avoiding the equilibration of magnetization, transient Overhauser experiments were performed [29]. This allows identifying the role of cross-relaxation in a straightforward manner, and clearly reveals the mechanism underlying the intensity enhancement when comparing spectra recorded with and without a π pulse [30, 31].
As shown in Fig. 2.2 for the microcrystalline proteins GB1 and ubiquitin, significant enhancements of some signals are observed after cross-relaxation delays tcr of a few hundreds of milliseconds.
Promoting uniform enhancements with low-power PARIS irradiation
In analogy to solution-state NMR, as illustrated in Fig. 2.5 for L-threonine, significant variations of NOE enhancement factors from one carbon site to another constitute a major impediment to a quantitative utilisation of 13C peak intensities in transient NOE spectra. To record quantitative spectra, the magnetization needs to be equilibrated. . As shown in Fig. 2.5, this can be accomplished by PARIS irradiation that simultaneously promotes heteronuclear Overhauser enhancements and the equilibration of magnetization. Utilizing this dual benefit from PARIS irradiation, the spectrum with uniformly enhanced peak intensities that are proportional to the number of nuclei was obtained (fig. 2.5(right)).
Studies on mitochondrial membrane protein TSPO
TSPO is a membrane protein that was previously named Peripheral-type Benzodiazepine Receptor (PBR) because of the binding of diazepam, a well-known benzodiazepine, which was initially observed in the kidney. The 18-kilodalton translocator protein TSPO is found in mitochondrial membranes and mediates the import of cholesterol and porphyrins into mitochondria. TSPO transports cholesterol through the external mitochondrial membrane and transfers it to the inner membrane with the assistance of the outer mitochondrial membrane voltage-dependent anion channel (VDAC) and ATPase family AAA domain-containing protein 3 (ATAD3A), which is an integral MP of the inner mitochondrial membrane crossing to the outer membrane [34]. In line with the role of TSPO in mitochondrial function, TSPO ligands are used for a variety of diagnostic and therapeutic applications in animals and humans. The three-dimensional high-resolution structure of mammalian TSPO reconstituted in detergent micelles in complex with its high-affinity ligand PK11195 was recently deciphered [35]. The TSPO-PK11195 structure is described by a tight bundle of five transmembrane α-helices that form a hydrophobic pocket accepting PK11195. Ligand-induced stabilization of the structure of TSPO suggests a molecular mechanism for the stimulation of cholesterol transport into mitochondria.
Table of contents :
Remerciements
1Overview of solid state NMR
1.1 Introduction
1.2 Dipolar spin interactions in solid state NMR
1.3 Basic experimental techniques: Magic-Angle Spinning (MAS) and Cross
Polarization (CP)
1.4 Dipolar recoupling and decoupling
1.5 Sensitivity enhancement techniques
1.6 Introduction to internal dynamics in biomolecules
1.7 Heteronuclear Overhauser Effect in organic solids
1.8 References
2 Sensitivity enhancement and quantitative 1D & 2D 13C spectra
2.1 Introduction to quantitative solid state NMR
2.2 Results and discussion
2.2.1 Overcoming the T1 (13C) constraints
2.2.2 Revealing the sources of the heteronuclear Overhauser enhancement .
2.2.3 Promoting uniform enhancements with low-power PARIS irradiation
2.2.4 Conclusions
2.3 References
3 Site-specific heteronuclear Overhauser measurements in filamentous Pf1 macromolecular assembly
3.1 Introduction
3.2 Sample preparation
3.3 Results and discussion
3.3.1 Recording site-specific one- and two-dimensional 13C NOE spectra
3.3.2 Probing internal dynamics of methyl groups in Pf1
3.4 References
4 Probing the gel to liquid-crystalline phase transition and relevant conformational changes in liposomes by 13C magic-angle spinning NMR
4.1 Introduction
4.2 Materials and methods
4.3 Results and discussion
4.3.1 Probing trans-gauche conformational changes
4.3.2 Revealing chain melting temperature (Tm)
4.3.3 Conclusions
4.4 References
5 Determination of sample temperature in unstable static fields by combining solid state 79Br and 13C NMR
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussion
5.4 Conclusions
5.5 References
6 Sensitivity improvement during heteronuclear spin decoupling in solid-state NMR
6.1 Heteronuclear dipolar decoupling in solid state NMR
6.2 Experimental: NMR experiments and numerical simulations
6.3 Results and discussion
6.4 Conclusions
6.5 References
7 Effect of inherent rf field inhomogeneity on heteronuclear decoupling in solidstate NMR
7.1 Introduction
7.2 Experiments and numerical simulations
7.3 Results and discussion
7.4 Conclusions
7.5 References
