In the transferred NOESY experiment, a 2D 1H–1H NOESY spectrum is recorded on a sample of low molecular weight compounds in the presence of a substoichiometric amount of a protein. Since small molecules tumble rapidly in solution, the dominant 1H relaxation mechanisms during a NOESY experiment leads to weak positive NOESY peaks. By contrast, the ligands have a long correlation time when bound to the protein. In the case of rapid exchange between free and bound states (namely when the dissociation rate constant koff is greater than the longitudinal relaxation time T1, koff >>1/T1), this translates into very strong negative transferred NOEs in the 2D NOESY spectrum of the free ligand. In a mixture of compounds in the presence of a protein, the compounds that bind to the protein will exhibit strong negative NOEs, whereas non-binders will show very weak positive NOEs.
If two ligands bind simultaneously with similar residence times in adjacent sites on the protein surface, forming a ternary complex. In such a case, strong negative ligand–ligand NOEs can be observed: ILOE.
If two ligands bind competitively to the same protein binding pocket (and not simultaneously to adjacent hot spots) and if they exhibit similar residence times, NOE can be observed: INPHARMA
The mixing time is an important parameter in NOESY-based experiments. Transferred NOESY experiments typically require mixing times of 200–600 ms. For observation of ILOEs, the mixing time needs to be even longer (600–800 ms), whereas INPHARMA peaks have been observed at 70 ms. The optimum ligand-to-protein ratio ranges from 10:1 for small proteins to more than 100:1 for large proteins, with the protein concentration in the micromolar range.