TrNOE

Other enzyme-substrate or inhibitor interaction studies80 82 have been addressed, using a combination of STD and trNOE NMR experiments, in order to collect details on the substrate bound conformation (ligand perspective). In other cases, the availability of a labelled protein receptor83 have permitted to follow the induced chemical shift variations of the protein resonances upon ligand addition to the NMR tube by HSQC methods (protein perspective). 

In the transferred NOE (trNOE) experiment, a ID or 2D NOESY experiment is recorded [31]. The intermolecular NOE build-up to be used as a parameter in Eq. (5) arises from the bound state but is observed via the free ligand, requiring rapid exchange between bound and free states. The technique is described in more detail in Chapt. 16. 

Fig. 16.2 A simplified scheme of the trNOE concept. The ligand L in the free state has negligible cross-relaxation between protons Hi and H2 because of its rapid tumbling motion. Upon binding to the much slower tumbling protein 7 becomes effective and leads to a transfer of magnetization from Hn to H2. Because of the dynamic equilibrium the ligand is released back into solution where it is still in the magnetization state corresponding to the bound form. The same concept is also applicable to trCCR and trRDC (see Sects. 16.4 and 16.5).

Alternatively, the much more common situation in trNOE studies involves fast exchange on the chemical shift time scale where the observed resonance shifts are weighted averages of the corresponding shift in the free and bound state [13]. A full account of the complete relaxation matrix and conformational exchange effects for n spins has been performed by London et al. [13], and a similar treatment was later incorporated into the programme CORCEMA [14]. 

Fig. 16.3 The calculated trNOE intensity as a function of mixing time without (left part) and with (right part) spin diffusion through intermole-cular cross-relaxation. For the calculations the distance between the protons was assumed to be 2 A, and the rotational correlation time used was 0.1 ns for the free ligand and 10 ns for the pro-...

Another complication of the trNOE method may be the possibility that the ligand interacts nonspecifically with the protein. This has been discussed by Rao and coworkers [23]. Specificity of the interaction can best be demonstrated by a competition experiment with a strong binder. 

A nice example of an indirect structure determination using the trNOE method is the study of the conformation of a loop of the membrane protein bacteriorhodopsin (BR) [28]. Antibodies were raised against BR, and subsequently the complex of a heptapetide derived from BR was studied in complex with the antibody by trNOE. The bound conformation is a reasonably good representation of the conformation of the peptide in its native state in BR. 

In the case of a ligand that is weakly bound to a macromolecule in the fast exchange regime, the CCR rates are averaged with the population of free and bound conformation in analogy to the previously described trNOE (Sect 16.3) [50, 51]. 

In the situation where the interaction is weak, one of the traditional methods that can be applied to obtain structm-al information (internuclear distances) of the bound ligand is the so-called transferred NOE (trNOE) method. Recently, it became possible to use transferred cross-correlated relaxation (trCCR) to directly measure dihedral angles. The combined use of these two techniques significantly improves the precision of the structure determination of ligands weakly bound to macromolecules. 

Keywords Cross-correlated relaxation IKK/3 NEMO NMR trCCR trNOE... 

IKK I/cB kinase NBD NEMO binding domain NEMO NF/cB essential modulator NOE Nuclear Overhauser elfect RDC Residual dipolar coupling SAR Structure-activity relationship trCCR Transferred OCR trNOE Transferred NOE... 

Recently, we studied by trNOE and trCCR the interaction of a peptide derived from lKKj8 which is reported to interact with NEMO by trNOE and trCCR. The experimental data as well as the strategy to obtain the backbone torsion angles of this peptide is presented here in detail. 

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