Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to ga lectin-3, a mammalian lectin of 26 kD size, is studied to map the binding s ites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the N-15 heteronuclear single quantum coheren ce (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurem ents to a more quantitative determination of distances between discrete pro tein amide protons and the bound spin label is then described. This protoco l is discussed as part of a drug design strategy in which subsequent pertur bation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the prima ry ligand. This approach is novel in that it bypasses the need for structur e determination and resonance assignment of the target protein.