Cross-correlated relaxation-enhanced

Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation enhanced H- C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J Am Chem Soc 125 10420-10428... 

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. 

By combining the quantitative approach [23] to extract cross-correlated relaxation with resolution enhancement methods using restricted coherence transfer in a so-called forward directed TOCSY [27], Richter et al. could determine the ribose sugar conformation for all but two residues in a uniformly 13C,15N labeled 25mer RNA [28] and compare them to 3J(H, H) values determined using a forward-directed HCC-TOCSY-CCH-E.COSY experiment [29]. 

In this section, I review the work on nuclear multi-spin relaxation phenomena - the work where one of the involved spins belongs to electron will be covered in section 2.7. We begin with the cross-relaxation (nuclear Overhauser enhancement, NOE) measurements and continue with experiments designed for saturation transfer difference (STD) measurements. Next, we turn to investigations of more complicated multispin relaxation phenomena such as cross-correlated relaxation. Finally, papers devoted to relaxation-optimized methods and to large spin systems are also included in this section. 

Fig. 4. Pulse sequences for determining spin-spin relaxation time constants. Thin bars represent 7t/2 pulses and thick bars represent tt pulses, (a) the CPMG sequence, (b) the spin-lock sequence used for determining T p and (c) a two-dimensional proton-detected INEPT-enhanced CPMG. T is the waiting period between individual scans. The pulse train during the T period is used for suppression of cross-correlation effects, and the delay S is set to < (1/2)J. The delay A in (c) is set to (1/4)Jih and A is set to (1/4)Jih to maximize the intensity of IH heteronuclei and to (1 /8) Jm to maximize the intensity of IH2 spins. The phase cycling in (c) is as follows i = y,—y 2 = 2 x),2 —x), i = 8(x), 8(—x) Acq = x, 2(—x), x, —x, 2(x), —x, —x, 2(x), —x,x, 2(—x), x. The onedimensional version the proton-detected experiment can be obtained by omitting the ti delay.

The successful observation of NOESY correlations can depend critically on the choice of two parameters the mixing (t ) and repetition times, both of which depend on the spread of H in a molecule. Recommended RT s are about 2T for small to intermediate-sized molecules. They should be set to 1.2-1.8 s for small molecules (MW 400-450), 0.9-1.2 s for intermediate-sized molecules (MW 500-750), and, more conservatively, to 2-3 s when the T s are unknown. The choice of mixing times also is important, because if is too short, NOE enhancements do not have a chance to develop to detectable intensities, and NOESY cross peaks are not observed. On the other hand, if is too long, the NOE enhancements disappear because of relaxation, and, again, cross peaks are absent. Compromise mixing times should be about the average T value and can be set to 0.3-0.6 s for MW 400-750 and 1-2 s for very small molecules. 

The elucidation of the scalar coupling network by the correlation experiments is, apart from small molecules, not sufficient for the unambiguous, sequential and stereo-specific assignment. The complementary information of spatially adjacent protons is obtained via cross-relaxation experiments, the laboratory-frame nuclear Overhauser enhancement spectroscopy (NOESY) and the rotating-frame nuclear Overhauser effect spectroscopy (ROESY). These experiments provide also the distance restraints for the structure determination and help to recognize exchange processes. 

---