Sequential assignment
As correlation spectra (1H!) do not couple or connect different amino acids, the sequential assignment in homonuclear NMR relies on NOE cross peaks alone. Several NOEs can usually be observed between sequential residues, as these distances are close (but in a conformation dependent manner) and are used to establish the neighborship relations among the spin systems/ amino acids identified above. Primarily used are sequential alpha to the next amide - cross peaks, and sometimes NOEs between amide protons of sequential residues.
Nomenclature: d Na intra residue alpha-amide proton distance
d aN (i,i+1) alpha-proton distance to amide of next residue
[d aN (i,i+2) , ditto, residues separated by two]
The sequential NOEs all involve amide protons due to the “assymmetric nature of the amino acids. As the amide protons is located near the left hand side of the aa, all sidechain/ Ca protons are roughly the center. Therefore, NOEs are generally observed from side chain to the following residue, but not to the previous one.
The CaH-NH cross peaks are found in a region called fingerprint region (4.5-6ppm, 7.5-10 ppm). Every amino acid gives rise to one intra residue peak in this region (with the exception of proline (0) and glycine (2). The sequential NOEs are usually much stronger than other (medium/ or long range NOEs), and thus can quite reliably be identified.
The sequential assignment strategy starts then at any amino acid/spin system, and uses the sequential NOEs to find the following residue in the sequence.
Starting points of the tracing can be any residues, ideally however, one tries
- unique dipeptide sequences, and so on.