NOESY heteronuclear-edited

They are most often combined with traditional two-dimensional experiments such as NOESY and TOC-SY to yield a three-dimensional experiment. For example, in the case of an HSQC-NOESY spectrum of a protein, two of the axes represent the heteronuclei such as and the protons which are directly attached to the nitrogen nuclei, while the third axis contains chemical shifts of protons which share an NOE effect with the amide proton. This offers a significant increase in resolution compared to a traditional two-dimensional NOESY. A large array of these types of three-dimensional, heteronuclear-edit-ed experiments have been designed to extract structural information in various situations. 

When combining isotope filtering/editing with coherence transfer steps to multidimensional experiments, then further size restrictions apply. For example, isotope edited / filtered H TOCSY or COSY experiments are generally limited to systems of <10 kDa, because of their sensitivity to T2 relaxation. In larger systems, heteronuclear correlation spectroscopy can be used for the correspondingly labeled component, while structural information about both the labeled and unlabeled moiety can be extracted from isotope edi-ted/filtered NOESY spectra, respectively. 

The most straightforward isotope-editing method for selecting protons bound to a heteronucleus and suppressing all others is the simple acquisition of a spectrum with an indirect heteronuclear dimension (in the literature the term isotope editing is often used as a synonym for these techniques). This can be accomplished by a simple 2D HMQC or HSQC shift correlation, or a more elaborate 3D technique including an additional NOESY or TOCSY step (3D X-edited NOESY/TOCSY etc.), or even 4D experiments with a second heteronuclear shift dimension [13, 14]. 

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.

In order to completely assign the NMR spectra, heteronuclear 3D-NMR experiments have been performed. Experiments essential for the backbone assignment are the N-edited NOESY, HNCACB and CBCA(CO)NNH. These spectra allow for nearly complete assignment of the backbone atoms of apoLp-III. 

As mentioned, non-invasive or passive techniques have been added to many multidimensional and multinuclear pulse sequences such as the 2D edited-HSQC up to more complicated 3D and 4D heteronuclear-based NOESYs. The basic principle is to use a relatively long selective pulse to manipulate water. 

Clearly the homonuclear and the heteronuclear experiments could be combined in the reverse order i.e. HSQC-NOESY and HSQC-TOCSY. The main advantage in these schemes relates to N-edited experiments in which the narrower amide proton spectral width is sampled during f and the full proton spectral width is collected during t. On the other hand water suppression is more effective when HSQC follows NOESY. Also the sensitivity enhancement can be incorporated into the NOESY-HSQC experiment. Eor the TOCSY-HSQC or HSQC-TOCSY it does not matter because both the TOCSY and HSQC sequences can be implemented with the sensitivity enhancement. It should be mentioned that the TOCSY type of transfer is more effective between C nuclei than between protons and therefore the HCCH-TOCSY experiment is preferred when a doubly labeled sample is available. 

Information on side-chain resonances or sequential connectivities can be obtained by converting the 2D-iH-i heteronuclear correlation experiment into a 3D experiment by adding either a TOCSY or a NOESY step. These 3D experiments can be considered as a series of 2D homonuclear experiments in which each is edited by a different frequency. Thus, having established amide H/i pairs via the HSQC, the TOCSY-HMQC correlates alpha protons... 

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