Chemical shifts are very sensitive to the electronic environment of a nucleus. Perturbations in the chemical shift can be caused not only by a change in the covalent molecular structure, but also through non-covalent interactions with solvent molecules or binding partners. This makes chemical shifts a very sensitive probe for the identification of interaction surfaces in protein complexes. Usually a series of experiments is recorded in which increasing amounts of a ligand or interaction partner are added to the protein of interest and the chemical shifts are monitored. Typically, a ¹H-¹⁵N HSQC spectrum is used since this is a highly sensitive experiment in which the peaks are generally well resolved and there is a probe for each (non-Proline) amino acid in the protein. As the interaction partner is added, some chemical shifts will be perturbed. Usually, these belong to amino acids close to the interaction surface. However, it is important to remember that a change in chemical shifts merely implies that there is a change in the magnetic environment of a nucleus, not a direct interaction with a binding partner. Thus, if a protein undergoes substantial structural rearrangement upon complex formation, widespread chemical shift perturbations may be observed, including in residues which are far from the interaction site (but which nonetheless experience a change in their structural environment).

Chemical shift mapping is best suited to weak interactions in the mM range (giving rise to fast exchange). In this situation the chemical shifts change continuously as the binding partner is added. Essentially, the interaction is so weak, that the complex only exists for part of the time it takes to record the NMR experiment. The observed chemical shift is therefore a weighted average of the chemical shifts for the free protein and the complex until the binding partner has been added to excess. If the binding interaction is stronger, it moves into the intermediate exchange regime in which protein resonance peaks may become weaker and finally disappear as their interaction partner is added. At high ligand concentrations the peaks may reappear (possibly at new positions). The ligand is now present in such excess that it forces all protein molecules into the complex form and the chemical shifts observed are those of the complex. Very strong binding interactions will be subject to slow exchange. As the ligand or binding partner is added some new resonance peaks will appear in the spectrum and grow in intensity while simultaneously some original resonances will gradually decrease in intensity until they disappear. The binding event is so strong that for the duration of the experiment any one protein molecule is either free or in complex and chemical shifts for both are observed simultaneously.

Chemical shift mapping is a very straight forward method and can provide information about both the location and strength of a binding event.