Proteins NOESY-HSQC

Note that analogous experiments, such as the 13C-edited HSQC—NOESY,72 can be performed on 13C-labeled proteins. For labeled proteins, this latter experiment provides the largest number of conformational restraints for protein structure calculations (see Section 9.09.5.2). The 15N-edited NOESY-HSQC only provides distance information for protons that are close in space to amide protons (since magnetization originates and/or terminates... 

The resolntion that is gained by the additional freqnency dimension (N in this case) is illnstrated in Figure 3.6. The two-dimensional NOESY spectrnm of a 50 residne a-helical protein is shown in the left part of this fignre. In this experiment, the magnetization is transferred from proton H to proton Hy by dipolar coupling. The first freqnency dimension corresponds to the chemical shift of H and the second frequency dimension corresponds to the chemical shift of Hy. As with the NOESY-HSQC described previously, the intensity of the peak is related to the distance between H and Hy. Note the large number of unresolved overlapping peaks in the... 

Muhandham DR et al (1993) A gradient 13C NOESY-HSQC experiment for recording NOESY spectra of 13C-labeled proteins dissolved in H20. J Magn Reson B 102(3) 317-321... 

More recent applications comprise, for example, the identification of the binding site of 18 kDa human cardiac troponin C for the drug bepridil [36]. For this study, the unlabeled ligands were bound to selectively [13CH3-Met, Phe-d8]-labeled protein (Fig. 17.8). First, the 13CH3-Met signals of troponin C were easily identified from 13C-HSQC spectra. In a 2D NOESY spectrum with 13C-editing in one dimension, intermolecular NOEs could... 

Fig. 17.8 Intermolecular NOEs between [13CH3-Met, Phe-ds]-labeled cardiac troponin C and the drug bepridil (left panels, drug protein 1.5 1 right panels, 3.5 1). A, D methyl region of the H spectrum B, E HSQC spectra showing the 13CH3-Met signals of the protein C, F section from the NOESY spectrum with 13C-editing in one dimen-...

In the following, three different experiments are discussed, where short, high-power spin-lock pulses are used to purge the spectrum from undesired resonances. The experiments are (i) the HSQC experiment [5], (ii) experiments with C half-filter elements [6], and (iii) NOESY and ROESY experiments for the observation of water-protein NOEs [7]. In the first two experiments, spin-lock purge pulses are used to suppress the signals from... 

Strip plots can only be constructed when the crosspeaks have already been assigned in the 2D HSQC spectrum. In a 15N-labeled protein, sequence-specific assignments come from sequential NOE (a,N, /3,N and N,N) crosspeaks located in the 3D HSQC-NOESY spectrum. The walk through the protein backbone is done in the same way as with unlabeled proteins, except that overlap in NOESY spectra is greatly reduced by spreading the crosspeaks out in the 15N dimension of a 3D spectrum. 

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.

Multidimensional NMR spectra are not restricted to cases where the separate frequency axes encode signals from different nuclear types. Indeed, much of the early work on the development of 2D NMR was performed on cases where both axes involved chemipal shifts. The main value in such spectra comes from the information content in cross peaks between pairs of protons. In COSY-type spectra (COSY = Correlation SpectroscopY) cross peaks occur only between protons that are scalar coupled (i.e., within 2 or 3 bonds) to each other, whereas in NOESY (NOE Spectroscopy) spectra cross peaks occur for protons that are physically close in space (<5 A apart). A combination of these two types of 2D spectra may be used to assign the NMR signals of small proteins and provides sufficient information on internuclear distances to calculate three-dimensional structures. Figure 12.3 includes a panel showing the COSY spectrum of cyclosporin and highlights the relationships between ID H-NMR spectra and corresponding 2D homonuclear (COSY) and heteronuclear (HSQC) spectra. 

The heteronuclear NMR experiments discussed above highlight how much extra resonance dispersion can be gained via this approach. The power of this added dimension becomes clear if, for example, the 3H—15N HSQC experiment shown above, where each HN atom is essentially resolved, was to be combined with a TOCSY or NOESY experiment to provide a third frequency dimension. The resulting 3D 15N-HSQC-TOCSY/NOESY spectrum would contain virtually no overlap of interresidue resonances. Such experiments are indeed possible and have been the driving force in producing uniformly 15N- and/or 13C-labeled proteins. This field has been the most intensely researched area of NMR in the past 20 years, and the strategies employed to determine protein and peptide structures using heteronuclear NMR experiments are discussed in the next section (see Chepter 9.19). 

A new element for simultaneous indirect detection of C and signals in labelled proteins was proposed by Uhrin et The CT- CHs, VT- N-HSQC sequence combines constant-time carbon evolution with variable delay nitrogen evolution. This is achieved by the variation of the position of a 90° pulse creating transverse coherence within the C constant-time period. The maximum indirect acquisition time for both nuclei is determined by constant-time period set to l/ /(C,C) = 28.6 ms. The method is best suited for detection of CH3 signals due to their slower relaxation. The proposed element was incorporated into NOESY-based experiments resulting in 3D NOESY-CH3NH and 3D HSQC-NOESY-CH3NH sequences. The experiments... 

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