Cross-peak determination

The HETCOR spectrum of a naturally occurring isoprenylcoumarin is shown in Fig. 5.41. The spectrum displays one-bond heteronuclear correlations of all protonated carbons. These correlations can easily be determined by drawing vertical and horizontal lines starting from each peak. For example, peak A represents the correlation between a proton resonating at 8 1.9 and the carbon at 8 18.0. Similarly, cross-peaks E and F show that the protons at 8 4.9 and 5.1 are coupled to the same carbon, which resonates at 8 114.4 i.e., these are the nonequivalent protons of an exomethylenic... 

The ROESY spectrum of podophyllotoxin exhibits a number of crosspeaks (A-D) representing interactions between dipolarly coupled (space coupling) hydrogens, which can be helpful to determine the stereochemistry at different asymmetric centers. For example, based on the assumption that the C-1 proton (8 4.53) is /3-oriented, we can trace out the stereochemistry of other asymmetric centers. Cross-peak B represents dipolar coupling between the C-1 proton (8 4.53) and the C-2 proton (8 2.8), thereby confirming that the C-2 proton is also... 

Considering all potential experimental and systematic errors of NOE/ROE crosspeak intensities, it is remarkable how robust the derived distance restraints still are. The reason Ues in the dependence of the cross-relaxation rate even if a cross-peak intensity is determined wrongly by a factor 2, the resulting distance restraint is only affected by the factor 1.12, which usually lies within the error range of distance restraints used in structure calculations. It should be further noted that the quaUty of a resulting structure is not so much determined by the... 

Fig. 9.4 The fixation of the Ala proton in cyclosporin A via ROE-derived distance restraints as an illustrative example for NMR structure determination. (A) The amide region of the 2D ROESY spectrum of cyclosporin A at room temperature with the cross-peaks to...

The hyperfine coupling tensors of carotenoids were determined from the HYSCORE analysis of the contour line-shapes of the cross-peaks (Dikanov and Bowman 1995,1998, Dikanov et al. 2000), which provided the principal components of the tensors that appear to be rhombic. Such tensors are characteristic of planar conjugated radicals with the unpaired spin in a pz orbital of the carbon of the C-H group. 

The cross-peak coordinates represent two frequency values, va and vp, where va + vp=2v, and v is the proton frequency. When plotted in the coordinates v2a and v2p, the contour lineshape is transformed into a straight line segment. An extrapolation of this straight line permits the determination of the hyperfine tensors. A curve obtained by choosing some frequencies in the range will intersect the line defined by the squares of the values v2a and v2p in two points. The values where the curve intersects the experimental data are (val, vpi) and (va2, vp2), where va=A/2 + v, and vp= Vj-A/2. This gives two values of the anisotropic coupling tensor, Ar... 

Another interesting possibility relies on the recording of two complementary IP and AP data separately and then added/subtracted to provide spin-state-selective a//1-HSQMBC spectra.119 In these types of IPAP spectra, the magnitude of "Jch can be directly determined by simple analysis of the relative displacement between a- and /1-cross-peaks. 

The last 90° pulse on 13C acts as a purge pulse for the undesired dispersive magnetization.47,48 The function of the pulse is to convert any magnetization remaining antiphase with respect to the 13C spin into unobservable multiple-quantum coherence. This will provide cross peaks with pure lineshapes and with higher resolution, and consequently establishes reliable determination of coupling constants.47,48... 

The quantification of an NOE amounts to determining the volume of the corresponding cross peak in the NOESY spectrum. Since the linewidths can vary appreciably for different resonances, cross-peak volumes should in principle be determined by integration over the peak area rather than by measuring peak heights. However, one should also keep in mind that, according to Eq. (1), the relative error of the distance estimate is only one sixth of the relative error of the volume determination. Furthermore, Eq. (1) involves factors that have their origin in the complex internal dynamics of the macromolecule and are beyond practical reach such that even a very accurate measurement of peak volumes will not yield equally accurate conformational constraints. 

In de novo three-dimensional structure determinations of proteins in solution by NMR spectroscopy, the key conformational data are upper distance limits derived from nuclear Overhauser effects (NOEs) [11, 14]. In order to extract distance constraints from a NOESY spectrum, its cross peaks have to be assigned, i.e. the pairs of hydrogen atoms that give rise to cross peaks have to be identified. The basis for the NOESY assignment... 

For each NOESY cross peak, one or several initial assignments are determined based on chemical shift fitting within a user-defined tolerance range. 

---