Relaxation inverse detection

Barbate G, Ikura M, Kay L E, Pastor R W and Bax A 1992 Backbone dynamics of calmodulin studied by N relaxation using inverse detected two-dimensional NMR spectroscopy the central helix is flexible S/oefrem/sf/ y 31 5269-78... 

For relaxation studies of biomolecules in solution (which is no specialty of the authors of this chapter), it is often essential to use inverse-detection schemes to obtain reasonable sensitivity. Furthermore, besides problems with poor sensitivity, the carbon-13 and nitrogen-15 spectra are often too crowded to allow measurement of individual relaxation rates for different I nuclei, either by direct detection or by indirectly detecting the protons. If this is the situation, one can spread out the I nuclei signals for better resolution of individual resonances by detecting a two-dimensional H-I correlation spectrum. Relaxation experiments of this type can be considered a modification of the double polarization-transfer IS correlation experiment [7, 17] ... 

As for inverse-detected one-dimensional relaxation measurements, there are problems related to the removal of proton signals originating from H not bound to C nuclei and to the fact that the H spectrum is in general... 

For larger molecules the situation is different because of the more complicated spectra and because of the smaller NOE enhancements. Two-dimensional inverse-detection experiments are usually the only practical solution to performing relaxation measurements in larger systems, such as biomolecules. In principle, it should be possible to obtain the same signal-to-noise ratio by two-dimensional and one-dimensional methods in... 

Indeed, paramagnetic broadening is proportional to the square of the nuclear magnetogyric ratio y. Therefore, the inverse detection of heteronucleus (i.e. the transfer of magnetization from the X nucleus to the proton followed by proton detection) may prevent the observation of X signal because of proton 72 relaxation. In fact, the relative values of y for H, 13C and, 5N nuclei are 1 1/4 1/10, and thus the relative contribution to overall relaxation arising from the hyperfine interaction is 1 1/16 1/100, respectively. Therefore, to identify... 

FIGURE 24. ll detected H—29Si correlation spectra of 15 ( II frequency 600 MHz, 5 mm inverse detection broad-band probe, relaxation time 2.0 s, acquisition time 0.38 s, 64 transient for each of 64 increments). (The two non-equivalent CH2 protons are distinguished by the letters a and b.) (a) Phase-sensitive spectmm measured with HSQC constant time experiment described in text (b) magnitude presentation of HMBC spectrum. Reproduced by permission of Academic Press from Reference 212... 

Dipolar Couplings and Distance Information. - The nuclear Overhauser effect (NOE) arises from dipolar interactions between magnetic moments associated with nuclear spins and it has become a powerful tool to extract relevant pieces of structural information about small molecules, as well as in molecules of biological interest. As a consequence, accurate NOE measurement is a very crucial issue. Walker et presented a comparison between direct and a new inverse HOESY experiment aimed at the detection of heteronuclear NOE between H and which is particularly well suited for symmetric compounds. It transpires that directly detected data are more suitable for quantitative assessment even if they suffer from lower sensitivity, whereas inverse detection is quite appropriate for a quick and quahtative assessment. In the latter experiment, unwanted cross-correlation effects may hide valuable NOE data (cross-relaxation), this drawback can be circumvented by a slight modification of the pulse sequence. 

All H NMR spectra were obtained with three signals suppression, i. e., water, methyl and methylene of ethanol signals in a 5 mm inverse-detection probe head. Eight FIDs were collected as 65536 data points using a 8.5 ps pulse (90°) spectral width, 8013 Hz acquisition time, 4.1 s and relaxation delay, 6.4 s. Spectra were processed using 32768 data points by applying an exponential line broadening of 0.3 Hz for sensitivity enhancement before Fourio transformation and were accurately phased, baseline adjusted and converted into JCAMP format to build the data matrix. 

The only platinum nucleus with magnetic properties is Pt, F=, (33.7% abundance). The resonance frequency in a magnetic field of 2.35 T is approximately 21.4 MHz. Satellite peaks from coupling with Pt were observed in H and P NMR spectra in the 1960s, and much of the early work on Pt detection used INDOR methods. Direct one-dimensional observation of Pt NMR spectra is now routine. Because Pt relaxation times are short for most compounds, there need be only a very short delay between pulses, allowing rapid accumulation. Two-dimensional inverse detection methods are also being increasingly used. 

Many optical studies have employed a quasi-static cell, through which the photolytic precursor of one of the reagents and the stable molecular reagent are slowly flowed. The reaction is then initiated by laser photolysis of the precursor, and the products are detected a short time after the photolysis event. To avoid collisional relaxation of the internal degrees of freedom of the product, the products must be detected in a shorter time when compared to the time between gas-kinetic collisions, that depends inversely upon the total pressure in the cell. In some cases, for example in case of the stable NO product from the H + NO2 reaction discussed in section B2.3.3.2. the products are not removed by collisions with the walls and may have long residence times in the apparatus. Study of such reactions are better carried out with pulsed introduction of the reagents into the cell or under crossed-beam conditions. 

Although long-time Debye relaxation proceeds exponentially, short-time deviations are detectable which represent inertial effects (free rotation between collisions) as well as interparticle interaction during collisions. In Debye s limit the spectra have already collapsed and their Lorentzian centre has a width proportional to the rotational diffusion coefficient. In fact this result is model-independent. Only shape analysis of the far wings can discriminate between different models of molecular reorientation and explain the high-frequency pecularities of IR and FIR spectra (like Poley absorption). In the conclusion of Chapter 2 we attract the readers attention to the solution of the inverse problem which is the extraction of the angular momentum correlation function from optical spectra of liquids. 

The idea of exploration of relaxation correlation was first reported in 1981 by Peemoeller et al. [23] and later by English et al. [24] using an inversion-recovery experiment detected by a CPMG pulse train. This pulse sequence is shown in Figure 2.7.1. 

Because the excitation/detection coil is in the x-y plane and the longitudinal component relaxes along the z axis, T cannot be measured directly from an NMR spectrum, but must be obtained using a pulse sequence. The most commonly used pulse sequence to measure T is an inversion recovery pulse sequence (Kemp, 1986). Other commonly used pulse sequences for measuring 7j are given in Ernst et al. (1987). 

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