Heteronuclear shift-correlation principle

The task of generating a display of heteronuclear X/Y-connectivities with optimum sensitivity can in principle be performed by recording a three-dimensional proton detected shift correlation in which the chemical shifts of both heteronuclei X and Y are each sampled in a separate indirect dimension. Three-dimensional fourier transformation of the data then generates a cube which is defined by three orthogonal axes representing the chemical shifts of the three nuclei 1H, X, Y, and the desired two-dimensional X/Y-correlation is readily obtained as a two-dimensional projection parallel to the axis... 

Figure 12.12a gives a good illustration of the need for going to a third dimension to facilitate the interpretation of a crowded 2D spectrum. The NOESY spectrum of a uniformly 15N-enriched protein, staphylococcal nuclease, has so many cross peaks that interpretation is virtually impossible. However, it is possible to use, 5N chemical shifts to edit this spectrum, as indicated in Fig. 12.121) and c in a three-dimensional experiment. With the 15N enrichment, NOESY can be combined with a heteronuclear correlation experiment, in this case HMQC, but HSQC could also be used. A 3D pulse sequence can be obtained from two separate 2D experiments by deleting the detection period of one experiment and the preparation period of the other to obtain two evolution periods (q and t2) and one detection period (f3). In principle, the two 2D components can be placed in either order. For the NOESY-HMQC experiment, either order works well, but in some instances coherence transfer proceeds more efficiendy with a particular arrangement of the component experiments. We look first at the NOESY-HMQC sequence, for which a pulse sequence is given in Fig. 12.13. The three types of spins are designated I and S (as usual), both of which are H in the current example, and T, which is 15N in this case.

Both types of experiments detect magnetization arising from mutually coupled heteronuclear spin-triples. Even if the spectra can in principle be displayed in a 3D-cube representation (Rg. 10), the analysis of 2D slices or projections is often preferred for the interpretation. Important features can be derived as is demonstrated in the example shown in Fig. 10, e.g. from inspection of H,C planes taken at the chemical shift of an individual phosphorus atom, which give a two-dimensional H,C correlation of all signals... 

In principle, a single three-dimensional correlation spectrum of an oriented sample of a uniformly N labelled protein provides sufficient information in the form of orientationally dependent frequencies for each amide site to determine the complete structure of the polypeptide backbone. The two-dimensional iH-i-TsT heteronuclear dipolar- N chemical shift PISEMA spectrum in Figure 5A was obtained from a uniformly i-TsT labelled sample of the 50-residue fd coat protein in oriented bilayers it contains resonances from all of the amide backbone (and side-chain) nitrogen sites. The resonances in the box in the upper left are largely from residues in the... 

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