Detection of protein-ligand NOEs with small, weakly binding ligands by combined relaxation and diffusion filtering

The use of a diffusion filter is proposed to suppress the NMR signals of small organic compounds in the presence of macromolecules. Combined with a spin-echo relaxation filter, the diffusion filter enables the selective and simultaneous detection of intermolecular solvent-protein NOEs in a straightforward two-dimensional NOESY experiment. Using the intermolecular NOEs observed between N,N-dimethyl-formamide (DMF) and hen egg-white lysozyme in an aqueous solution containing 2 M DMF, the binding of DMF at the specificity-determining substrate binding site C of the enzyme was modelled.