Heparin and heparan sulfate (HS) glycosaminoglycans (HSGAGs) are sulfated polysaccharidesthat play important roles in fundamental biological processes by binding to proteins. The prototypic exampleof HSGAG-protein interactions is that with the fibroblast growth factors (FGFs), specifically FGF1 andFGF2. Structural and biochemical studies have shown that the chain length, sulfation pattern, andconformation of HSGAGs play a critical role in FGF binding and activity. Previously, we showed that atetrasaccharide of the form ANS,6X-I2S-ANS,6X-I2S-OPr (where X is OH or O-sulfate and Pr is propyl) withat least one of the ANS,6X residues having a 6-O sulfate group was the minimum binding motif for FGF1[Guerrini, M., Agulles, T., Bisio, A., Hricovini, M., Lay, L., Naggi, A., Poletti, L., Sturiale, L., Torri, G.,and Casu, B. (2002) Biochem. Biophys. Res. Commun. 292, 222-230]. We report NMR structural analysisusing two-dimensional NOE spectroscopy (2D-NOESY) and transferred NOESY (trNOESY) on a non-6-O-sulfated synthetic tetrasaccharide TETRA (ANS-I2S-ANS-I2S-OPr) both in its free state and bound toFGF2. This tetrasaccharide comprises both the structural trisaccharide motif ANS-I2S-ANS that forms "kinks"in longer heparin chains induced by FGF binding [Raman, R., Venkataraman, G., Ernst, S., Sasisekharan,V., and Sasisekharan, R. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2357-2362] and the common bindingmotif I2S-ANS-I2S present in octasaccharides that exhibited strong FGF2 binding [Kreuger, J., Salmivirta,M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744-30752]. Thesedata suggest that TETRA could be the shortest HSGAG oligosaccharide that binds to FGF2. Furthermore,our study confirms that both the IdoA residues in TETRA adopt the chair 1C4 conformation upon FGF2binding to provide the best molecular fit in contrast to an analogous 6-O-sulfated tetrasaccharide motifobserved in the FGF2-HSGAG cocrystal structure where one of the IdoAs adopts skew-boat 2SOconformation. Thus, our study highlights the fact that the conformational plurality of IdoA is able toaccommodate the changes in the sulfation pattern to provide the necessary specificity for protein binding.