Assignment of homonuclear spectra

The principle process of homonuclear sequential assignment was developed by Kurt Wüthrich and coworkers. Experiments as 2D COSY and TOCSY are employed for the identification of amino acid spin systems (blue arrows). The 2D NOESY experiment is used to sequentially connect the spin systems (red arrows).

The fist step in sequential assignment is the identification of certain amino acids, with a characteristic pattern of cross signals, i.e. of glycine, alanine, threonine, valine, leucine and isoleucine.

Glycine (left picture) contains two H^alpha protons and is therefore readily identified. Valine (right picture), leucine and isoleucine can be recognized by their two methyl groups which give a characteristic row of double signals between 0 and 1.5 ppm. In the same way, alanine and threonine are identified by their single methyl groups.

In the second stage of the assignment process, the sequential contacts from the already identified amino acids to the neighboring ones are searched for in the 2D NOESY spectra. The connectivity of a given amino acid in the sequence (i) to its following one (i+1) can be observed in the NOESY because the distance of the amide proton of (i+1) to the H^alpha, H^beta or H^gamma protons of (i) is smaller than 5 A in almost every case (left picture). Therefore, sequential cross signals to H^alpha(i), H^beta(i) etc. are observed at the frequency of H^N(i+1) (right picture, dark blue signals). These interresidual cross signals can be distinguished from the intraresidual ones by comparing the 2D NOESY with the 2D TOCSY spectrum. A series of these sequential cross signals between H^alpha(i) and H^N(i+1) determines the order (i, i+1, i+2,...) of the amino acid spin systems.
Thus, dipeptides are identified and subsequently prolonged to oligopeptides by the search for further sequential contacts. Some time along the line these oligopeptides can be placed at a unique place in the primary structure by comparison with the amino acid sequence of the protein - they are sequentially assigned.
The chain of sequential connectivites is interrupted by proline residues because these have no amide proton. Therefore, no H^N(i)-H^alpha(i-1) cross signal can be observed. However, if the proline (i) is in itstrans conformation, the sequential H^N(i-1)-H^delta(i) and H^alpha(i-1)-H^delta(i) cross signals can be observed.
Another problem is, that this approach of sequential assignment breaks down for larger proteins because the vast number of signals leads to spectral overlap which hinders the identification of signals.