Cross relaxation-enhanced polarization transfer

With the adaptation of NMR techniques for larger molecules, it becomes possible to analyze proteins with molecular weights reaching 50 kDa. These techniques include H-, N-TROSY (transverse relaxation-optimized spectroscopy, with the mutual cancellation of H-, N-dipole-dipole coupling and the N chemical shift anisotropy) and CRINEPT (Cross-correlated Relaxation-Enhanced Polarization Transfer, combining insensitive nuclei enhanced by polarization transfer (INEPT) transfer with cross-correlated relaxation-induced polarization transfer). They are used in conjunction with the N-, c-labeling of the protein for increased sensitivity. 

TROSY (transverse relaxation-optimized spectroscopy) and CRIPT (cross-correlated relaxation-induced polarization transfer) or CRINEPT (cross-correlated relaxation-enhanced polarization transfer) for the two-dimensional (2D) NMR analysis of N-. H-labeled homo-oligomeric macromolecules with masses ranging from 110-800 kDa. Practical applications of these methods are, for instance analyses of intermolecuiar interactions in supramolecular complexes or conformational changes of a single macromolecule upon interactions with other molecules. 

The majority of double-resonance solid-state NMR experiments involving spin-1/2 nuclei use transfer of nuclear polarization via dipolar cross polarization (CP) to enhance polarization of the diluted spins S with small gyromagnetic ratio ys and significant longitudinal relaxation time T at the expense of abundant spins I with large y, and short 7 [215]. Typically, CP is used in combination with MAS, to eliminate the line broadening due to CS A, as well as with heteronuclear decoupling. To achieve the / S CP transfer, a (n/2)y pulse is applied at the I spin frequency,... 

Hence, provided that I g is known and that R has been determined by means of an independent experiment, provides the cross-relaxation rate ct. This enhancement is called nuclear Overhauser effect (nOe) (17,19) from Overhauser (20) who was the first to recognize that, by a related method, electron spin polarization could be transferred to nuclear spins (such a method can be worked out whenever EPR lines are relatively sharp it is presently known as DNP for Dynamic Nuclear Polarization). This effect is usually quantified by the so-called nOe factor p... 

Signal enhancements were obtained in H — O cross-polarization experiments without spinning, and reliable second-order quadrupolar powder patterns were observed [1291. Relaxation parameters involved in cross-polarization transfer were shown to be characteristic of the various sites, so that they can be used for signal assignment. In addition, in some cases the differences in crosspolarization rates were used to edit spectra by a selective enhancement of protonated oxygen resonances, such as those from surface hydroxyl groups in amorphous silica. The latter method can be applied to complicated systems, provided dipolar H — O interactions for the various sites are different. We illustrate this procedure by using a static H — O CP spectrum [126] of talc. 

Polarization transfer has also been observed between HP supercritical xenon and organic solutes. HP xenon was collected as a solid and then transferred to a 3 mm borosilicate tube containing the organic molecule (toluene or biphenyl) at a field of 1 T to avoid relaxation. Proton enhancements were observed to be three (biphenyl) or seven (toluene) times the Boltzmann equilibrium level as measured at 2T and with the xenon polarization at 2%. Several proton acquisitions could be made because the xenon Ty was approximately 7.5 min. The authors suggest that the low cross-relaxation rates observed in both the liquid and now supercritical phases could lead to better polarization transfer in the solid state, albeit with a highly dispersed xenon, such as provided by freezing a supercritical or liquid solution. 

The method, usually called cross-polarization (CP) C NMR, has several advantages over the conventional technique (Miknis et al., 1981 Smith and Smoot, 1990). The weak signals from the C atoms (abundance 1.1%) are enhanced by transferring polarization from protons to carbons and then decoupling the C-H couplings by irradiation with high-power radiofrequency. This enables the use of the short proton relaxation time to be used instead of the longer relaxation time as the... 

Hyperpolarized noble gas atoms, like xenon, can interact with protons and other nuclei via dipolar cross-relaxation, a mechanism which is supported by stochastic processes of motion. By means of this interaction, hyperpolarized xenon can transfer its polarization to protons and other nuclei of interest. An efficient way to make use of this SPINOE mechanism is to freeze hyperpolarized xenon onto the surface of the sample. During the defrosting process large amounts of xenon can penetrate into the liquid sample, resulting in a large SPINOE enhancement. 

This technique involves transfer of polarization from one NMR active nucleus to another [166-168]. Traditionally cross polarization (CP) was employed to transfer polarization from a more abundant nucleus (1) to a less abundant nucleus (S) for two reasons to enhance the signal intensity and to reduce the time needed to acquire spectrum of the less abundant nuclei [168]. Thus CP relies on the magnetization of I nuclei which is large compared to S nuclei. The short spin-lattice relaxation time of the most abundant nuclei (usually proton) compared to the long spin-lattice relaxation time of the less abundant nuclei, allows faster signal averaging (e.g., Si or C). CP is not quantitative as the intensity of S nuclei closer to 1 nuclei are selectively enhanced. Nowadays CP has been extended to other pairs of... 

Isotopes in low abundance have long spin-lattice relaxation times which give rise to poor signal-to-noise ratios. Sensitivity can be improved by using a technique known as cross polarization where a complex pulse sequence transfers polarization from an abundant nucleus to the dilute spin thereby enhancing the intensity of its signal. 

The benefits are primarily an intensity enhancement of the dilute spin signal by a factor of 7 abundant/7 dilute and a reduction of the recycle time between experiments since the ratedetermining relaxation time is now that of the abundant species, rather than that of the rare spins. Usually, the relaxation of the abundant spins are much faster than the dilute spin relaxation (13). The cross polarization experiment may thus be repeated with much shorter intervals, leading to a further increase of the signal-to-noise ratio of the rare spin NMR spectmm within a given period of time. The effectiveness of the cross polarization experiment depends on the strength of the dipolar interaction between the abundant and rare spin systems, i.e. on the distance between the actual nuclei (proportional to r, where r is the distance between the abundant and the dilute nuclei) (11). The efficiency of magnetization transfer decreases extremely fast as the distance between the abundant and rare spins increase. One should emphasis, that under normal conditions, the CP experiment does not provide quantitative results. Finally, the cross polarization sequence does not influence the line width. 

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