Assignment Theory

The most simple and straight forward method of backbone resonance assignment involves the use of 15N,13C labelled protein and the measurement of CBCANNH and CBCA(CO)NNHspectra.

Large Proteins

Large proteins give worse NMR spectra, because they tumble more slowly. For this reason the CBCANNH and CBCA(CO)NNH spectra of larger proteins (> 150 residues) are often not of sufficient quality to be able to carry out a full assignment. In this case a good option is the use of HNCA, HN(CO)CA, HNCO and HN(CA)CO spectra.


For very large proteins (typically > 250 residues) it may become necessary to deuterate the protein. In this case varients of the CBCANNH and CBCA(CO)NNH are used (so-called out-and-back experiments) which are less sensitive, but due to the better relaxation properties of the protein the spectra may none-the-less improve.

Field strength

In principle it is good to move to higher fields (750 MHz or more) with larger proteins, since it is then possible to use TROSY techniques and the greater resolution helps when the degree of overlap is large. However, it is worth noting that the CBCA(CO)NNH or HN(CO)CA are likely to decrease in quality due to the increased effect of the carbonyl chemical shift anisotropy. So even for proteins of 200 or more amino acids it may prove better to record 3D data sets at 600 MHz.