Characterizing how ligands bind to proteins by NMR

Finding the approximate locations of binding sites can be done rapidly by nuclear magnetic resonance spectroscopy. Determining binding-site location by comparing the NMR chemical shifts of the free protein with those of the protein bound to a ligand is a standard technique. Those portions of the protein that show changes in chemical shifts in the bound form are likely to be near or in the binding pocket. The alteration in the molecule perturbs the chemical shifts of protein residues near that part of the molecule in the protein-ligand complex. With a series of structurally related ligands, the technique allows an approximate mapping of the ligand in the binding site. When the researchers replace the ethyl group in ascomycin with an allyl moiety, which yields FK 506 itself, no change in chemical shifts occurs. The information is not as detailed as that from structures obtained by X-ray crystallography or NMR.