3D NOESY

Fipire 6.8 Volume chosen for a selective 3D NOESY-TOCSY spectrum of a peptide with NH region in coi, in >.2, and all protons in [Pg.354]

D-NOESY- H- N-HMQC/HSQC Sequential assignment of the spin system, identification of nOes with amide protons... 

Identi6cation NOESY-NOESY NOESY-HMQC 3D NOESY-HMQC... 

Different assignment strategies can be employed, depending on whether selective or nonselective pulses have been used in recording 3D spectra. A homonuclear 3D NOESY-TOCSY spectrum in which the NH/ region has been recorded is presented in schematic form in Fig. 6.9. 

Another way of avoiding overlaps seen in two-dimensional spectra is to introduce the third dimension. This has been illustrated with the 3D NOESY-HOHAHA [21-25], 3D HOHAHA-NOESY [25-27], NOESY-COSY [28, 29], COSY-NOESY [28] and ROESY-TOCSY [23] experiments. A principal drawback, associated with homonuclear 3D techniques, is the low digital resolution achievable along the first and second spectral axes. This limitation can to certain extent be removed by implementing band selective pulses into the 3D homonuclear experiments [21, 26, 28-31]. 

An obvious extension to 3D spectroscopy from 2D spectroscopy is the homonu-clear NOESY-NOESY [34]. There are two t variable times and one tj, which after Fourier transform provide three frequency domains. The 3D NOESY-NOESY spectrum of met-myoglobin cyanide, which contains low spin iron(III) in a heme moiety (see Fig. 5.7), has been successfully measured [35]. In Fig. 8.22 a slice of the 3D spectrum is shown at the I2-CH3 height. On the diagonal it shows all the dipolar connectivities between I2-CH3 and other protons off-diagonal... 

Fig. 8.22. Cross-section of the 600 MHz 3D NOESY-NOESY spectrum of met-myoglobin cyanide. The slice is taken at the 12-CH3 height. The inset shows the simulated cross-section involving the 12-CHj, 13-Ha and 13-Ha signals [35],...

Results similar to those shown in the slice of Fig. 8.22 can be obtained with the so-called NOE-NOESY sequence [36]. Here a hyperfine shifted signal, e.g. I2-CH3 of the above compound, is selectively saturated, and then the NOESY pulse sequence is applied. The NOESY difference spectrum obtained by subtracting a NOESY spectrum without presaturation of the I2-CH3 signal is shown in Fig. 8.23. Here, some more cross peaks are evident with respect to the 3D NOESY-NOESY experiment because secondary NOEs develop much more when the primary NOEs from the I2-CH3 signal evolve in a steady state experiment like the NOE-NOESY rather than in a transient-type experiment like the NOESY-NOESY. In Fig. 8.23, dipolar connectivity patterns are apparent among protons... 

Figure 15 2D H, H projection of the mi -filtered 3D NOESY H, C HSQC spectrum that contains only intermolecular NOEs between the...

Concatenation of the 3H—15N HSQC (or HMQC) sequence with a JH—JH NOESY gives rise to the 3D 15N-edited NOESY-HSQC (or 3D NOESY-HMQC) experiment.66-68 Here, two of the frequency dimensions represent the amide JH and 15N chemical shifts, while the third dimension provides information about the chemical shift of protons with which each amide proton is dipolar coupled (i.e., separated by <5.5 A). The spectrum is routinely viewed as narrow 2D (JH—JH) strips taken at the 15N chemical shift of each crosspeak in the JH—15N HSQC spectrum (see Figure 14). 

Figure 14 Comparison of 2D NOESY and 3D 15N-edited NOESY-HSQC spectra of a 41-residue peptide toxin from the Australian funnel-web spider Hadronyche infensa. A strip from the 2D NOESY spectrum is shown on the far left and it illustrates overlapping NOE correlations from three different amide protons (those of Trp13, Lys17, and Gly33). Fortunately, the 15N nuclei for these three amide groups have unique chemical shifts and hence they appear on different 2D planes in the 3D NOESY-HSQC experiment. Strips from these three planes are shown on the right, and they demonstrate that all of the NOE correlations are perfectly resolved in the 3D experiment.

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... 

Bernstein, R., et al., Computer-Assisted Assignment of Multidimensional NMR Spectra of Proteins Application to 3D NOESY-HMQC and TOCSY-HMQC Spectra, J. Biomol. NMR, 3, 245, 1993. 

The comparison of the NUS spectra with the reference shows that reduction of the number of acquired increments to 30% results in good quality spectra, comparable to the reference and reduction of measurement time to one third. This is highly significant considering the fact that typical US 3D NOESY spectra for proteins are... 

Fig. 18. Comparison of the 2D NOESY of rhodniin and a slice through the 3D NOESY. Especially in the aliphatic region, the overlap is unresol vable in the 2D whereas it can be nicely resolved in 3D.

However, tertiary-structure information cannot be derived from this information yet. NOESY Spectra need to be recorded and analyzed in order to obtain a sufficient amount of proton pairs that are close in space to build a structure. Assignment of NOESY spectra is still a time-consuming job, since there is a lot of overlap even in 3D NOESY spectra. Tools for a handy interpretation of the spectra exist that assist in assigning the NOESY cross-peaks and in bookkeeping of them. As an example, a sequence of strips through the 3D NOESY of the CaM/C20W spectrum is shown in Fig. 31. 

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