Saturation transfer difference NMR spectroscopy for identifying ligand epitopes and binding specificities.

We have studied the potential of saturation transfer nuclear magnetic resonance (NMR) experiments in different mode to screen compound mixtures for binding activity and to characterize binding epitopes on the ligand. We have developed a protocol based on the transfer of saturation from the protein to bound ligands which by dissociation is moved into solution where it is detected (Fig. 1; Mayer and Meyer 1999; Peters and Meyer). By subtracting a spectrum, where the protein is saturated from one without protein saturation, a spectrum is produced where only signals of the ligand(s) remain in the difference spectrum. The irradiation frequency is set to a value where only protein resonances and no resonances of free ligands are located. Usually, irradiation frequencies around 1.5 ppm are practical because no ligand resonances are found in this spectral region, whereas the significant line width of protein signals still allows selective saturation. If the ligands show no resonances in the aromatic spectral region, the saturation frequency may also be placed there.