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

¹ Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA
² Department of Chemistry, International Christian University, Tokyo, Japan 1818585
³ Department of Experimental Medicine, Lund University, Lund, Sweden S-22362

Reprint requests to: Dr. James H. Prestegard, Complex Carbohydrate Research Center, 220 Riverbend Rd., University of Georgia, Athens, GA 30602, USA; e-mail: jpresteg{at}ccrc.uga.edu; fax: (706) 542-6281.

The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the ¹⁵N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to idey protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation 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 primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.

Keywords: Nitroxide spin label; galectin-3; drug design; drug screening; distance mapping

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