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The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein-carbohydrate interactions known. Here, the GM1 pentasaccharide was cleaved into smaller fragments to determine the contribution of each key monosaccharide residue to the overall binding affinity. Displacement isothermal titration calorimetry (ITC) allows measurement of all key thermodynamic parameters, even for the lowest affinity fragment ligands.