Sequential NOEs

As mentioned above, which of the sequential NOEs d j, d j, and dj j is observed depends on the conformation of the backbone for the residues involved. Repetition of a particular type of connectivity for a sequence of amino acids often occurs in regions of regular secondary structure (19). For example a stretch of d jj-type NOEs is a signature of extended conformation, whereas a sequence of dj j -type NOEs is characteristic of helical conformation. Turns, on the other hand, are characterized by short, distinct patterns of dj j and d j connectivities. 

Once the sequential assignment is complete, the secondary structure of the protein can be determined from the secondary-structure specific chemical shifts of the 13Ca nuclei. Other nuclei, including the 13C/9 and the 13CO, also show characteristic chemical shifts that can be used to identify the secondary structure [55-60]. In addition, the secondary structure can also be assigned with the help of the identified secondary structure-specific sequential NOEs and coupling constants obtained from the HNHA experiment... 

Scheme 1 Peptide Sequence Sequential NOEs and Observable Through-Bond Couplings Useful for Sequential Assignment of the Peptide...

A short, three-residue walk through the NOESY spectrum is shown in Figure 12.25. Although there is a significant overlap in this region, two prominent sequential NOE peaks connect a Leu to a Val (Ha (Leu) = 4.89 to Hn (Val) = 8.88) and the same Val to a Lys... 

So far we have not learned anything from the NOE data except to assign all of the protons. This is an essential first step, but the real goal is to extract distances between specific pairs of protons from the NOE data. Sequential NOEs are not very useful because we already know that an amino acid residue is close in space to its nearest neighbor in the sequence. The real mother lode of the NOESY data consists of the medium-range (i -> i + 2, i —> i + 3 and i -> i + 4) and long-range connectivities. These define the secondary structure... 

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. 

A. Intermediate intensity sequential NOEs, another sign of a-helix,... 

Based on sequential NOE pattern and proton exchange rate, as well as Ha chemical shifl s, backbone O, T angles for well formed a-helices can be derived (Forman-Kay et al., 1991). For example, in the calculation of E. coli trp repressor solution structures, Zhao et al. (1993) used O = -65° 45° and = -35° 45° for residues that had three or more characteristic helical NOEs and/or a slowly... 

In contrast to NOEs, scalar coupling relies on localized direct interaction. They have therefore potentiality to define precise local structure information. They can also be used to obtain stereospecific assignments (e.g., for C J1 methylene protons) in combination with intra-residual and sequential NOEs.72... 

With the dissemination of multinuclear multidimensional NMR experiments, the automated NMR resonance assignment became feasible and indispensable, simultaneously.23,35,41,99 Typically, 10-20 individual multidimensional NMR spectra are measured for a large protein. The NMR-derived information quality definitely relies on the resonance assignment. The precious assignment has been based on and assessed by the sequential NOE continuity on the peptide sequence and consistency with the amino acid spin... 

Fig. 31. Strip plot of the three-dimensional NOESY- N/H-HSQC spectra for residues E7 to S17 of the CaM/C20W complex. Dashed lines indicate sequential NOEs between residues.

Figure 5 Sequential NOE connectivities in DNA between sugar HI and base protons (H6 in pyrimidines and H8 in purines). The intra- and inter-residual connectivities are marked in red and blue, respectively.

A. Intraresidue NOEs from A4H8 B. Sequential NOEs from A4H8 to A3Hi. 

Because the sequential NOE pattern of the repressor in the complex (Figure 2) shows clear helical connectivities for all of the six a helices, continuous stretches of 5 or more NOEs characteristic of an a helix were assigned typical dihedral angle constraints phi=-65( 45)A and psi=-35( 45)A in both the previous (i, 10) and present structural calculations. 

Figure 2. Sequential NOEs in the trp repressor-mtr ODNA complex.

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