Cross peaks, projections

Figure 5.65. 2D-Shift correlated spectrum of the tricyclodecane derivative, showing decoupling in the (Uj dimension through the use of the pulse sequence shown in Figure 5.64. The spectrum on the top of the diagram is the fully coupled spectrum in the right margin is the decoupled projection of the 2D spectrum. The area enclosed by the two vertical dashed lines corresponding to the multiplet for proton J contains cross peaks. Projection to the right from these cross peaks affords the various chemical shifts of protons to which proton J is coupled.

In principle, a de novo protein structure determination requires one round of 7 Candid cycles. This is realistic for projects where an essentially complete chemical shift list is available and much effort was made to prepare a complete high-quality input of NOESY peak lists. In practice, it turned out to be more efficient to start a first round of Candid analysis without excessive work for the preparation of the input peak list, using an slightly incomplete list of safely identifiable NOESY cross peaks, and then to use the result of the first round of Candid assignment and structure determination as additional information from which to prepare an improved, more complete NOESY peak list as input for a second round of 7 Candid cycles. 

FIGURE 10. Contour plot of two-dimensional nuclear Overhauser effect ll NMR (NOESY) of the protonated Schiff base of all-traos-retinal, in chloroform, with formate as the counterion. The intermolecular NOE cross-peak observed between H15 of the retinal and the counterion proton, at a mixing time of 0.4 s, is shown. Top trace f2 projection of the 2D NOE spectrum. Reproduced by permission of John Wiley Sons from Reference 36... 

In contrast to the basic "C detected experiment, and as a consequence of the final H detection, the 2D spectra obtained with HMQC or HSQC have a projection onto the F2 axis which corresponds to the normal H spectrum and includes all chemical shifts and all Jfi, couplings. The latter may give rise to rather broad cross peaks for extensively coupled protons. The projection onto the Fl axis corresponds to a normal C spectrum but with the quaternary carbons missing. With HMQC, but not with HSQC, cross peaks are additionally split in Fl by "J couplings. The HMQC and the HSQC experiment are usually performed in phase-sensitive mode, which, after proper phasing in both dimensions, allow peaks to be displayed in pure absorption. 

Use the stored ID spectra as projections and verify the correct calibration of the 2D spectrum. The selected peak(s) in the ID projection spectrum should appear at the same position (ppm) as the corresponding cross peak in the 2D contour spectrum. 

Heteronuclear NMR experiments are represented by the HSQC and HMBC spectra in Fig. FI. 4.6 and Fig. FI. 4.7. The 13C NMR spectrum (or 13C-projection) is displayed along one axis, and the H NMR spectrum (or -projection) along the other. The H-13C correlations are shown as cross-peaks in the spectrum. Contrary to homonuclear NMR experiments, there exist no diagonal and only one cross-peak for each correlation. 

Figure 8 31P 1H) HETCOR spectrum of OCP. For comparison, the H MAS spectrum is appended next to the H projection spectrum. The vertices of the rectangular box correspond to the cross-peaks of monetite (impurity phase). Note that the OCP signal also contributes to the cross-peak at — 0.2 ppm (3,P) and 13.6 ppm ( H). (Reprinted with permission from Ref. 102. 2004 Elsevier.)...

FIGURE 10.5 Application of the pulse sequence of Fig. 10.4 to dimethylformamide in DMSO-d6. The diagonal peaks from M, M2, and MF are indicated the other diagonal peaks arise from H20 impurity and DM SOU. (very weak). The cross peaks close to the diagonal result from methyl exchange the weaker peaks near (2.9, 8.0) are from the NOE. Shown at the top is a projection of intensity in the F2 direction. Courtesy of Herman J. C. Yeh (National Institutes of Health). 

Figure 32 Selected cross peaks and projections from the HMQC spectrum of Rh(PMe3)3Cl(H)2 obtained with para-B.2-(Figure courtesy of Prof S. Duckett)...

Figure 27. While the information content of the two presentations is identical, the stacked plot is related more easily to a conventional spectrum, and therefore used more widely when identification of individual peaks is of primary interest, as in J-spectroscopy. On the other hand, a contour plot is more economical when one is attempting to establish connectivities between atoms by an observation of cross peaks, as in COSY and NOESY spectra. Here a contour projection of the normal spectrum is plotted on the diagonal, and the cross peaks can be identified readily on the projections of their coordinates, as shown in Figure 26. The cross peaks in the COSY spectrum are indicative of spin-spin coupling between the two groups on the diagonal. The cross peaks in the NOESY spectrum indicate the existence of crossrelaxation between the two groups.

As a practical illustration. Fig. 5 shows 4 typical two-dimensional projections of the ten-dimensional spectmm of a small 39-residue protein agitoxin, globally enriched in and N. All these spectra were obtained by combining three- and four-dimensional experiments that were completed in a reasonably short time, whereas the duration of the full ten-dimensional experiment would have been completely unacceptable. These four planes have been selected from the full complement of 45 possible projections. Each of these spectra contains many cross-peaks because there are many different pairs of adjacent amino acids. 

Fig. 1 Illustration of the projection-cross-section theorem [17-19] for a 2D frequency space with two indirect dimensions k and j. ID data cf (t) on a straight line in the 2D time domain (ty, tj) (left) is related to a ID orthogonal projection (of) of the spectrum in the 2D frequency domain (

In a set of j projections with different projection vectors pi/, an Y-dimensional cross peak Q is projected orthogonally to the locations Qf Here,/is an arbitrary numeration of the set of j projections / = 1,. .., / In the 2D coordinate system of projection/ the projected cross peak has the position vector Ql = [v... 

The Al-dimensional cross peak Q is located in an (A — 2)-dimensional subspace, which is orthogonal to the projection plane at the point Q (Fig. 1). The peak subgroup of an A-dimensional chemical shift correlation Q is the set of projected... 

With these three mixing times, the 5D APSY-HC(CC-TOCSY)CONH experiment was recorded with a 1 mM solution of the 12.4-kDa globular protein TM1290 in 24 h of spectrometer time using 36 projections (Fig. 10). Based on the reference assignment of this protein, 424 cross peaks are expected in the resulting 5D APSY correlation peak list [43] 368 thereof were actually found in the present experiment. These 368 correlations contained the chemical shifts of 97% of the aliphatic carbons and 87% of the aliphatic protons in the protein. 

Plotting of the HMQC/HSQC data sets should include all cross peaks in lieu of the use of projections of the actual data matrix (with their poor digital resolution), we include 1-D spectra along the... 

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