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Distance mapping of protein‐binding sites using spin‐labeled oligosaccharide ligands
Author(s) -
Jain Nitin U.,
Venot Andre,
Umemoto Kimiko,
Leffler Hakon,
Prestegard James H.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.17401
Subject(s) - oligosaccharide , chemistry , binding site , plasma protein binding , biophysics , crystallography , computational biology , biochemistry , biology
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 15 N 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.