Residual dipolar coupling investigation of a heparin tetrasaccharide confirms the limited effect of flexibility of the iduronic acid on the molecular shape of heparin

The solution conformation of a fully sulfated heparin-derived tetrasaccharide, I, was studied in the presence of a 4-fold excess of Ca2+. Proton-proton and proton-carbon residual dipolar couplings (RDCs) were measured in a neutral aligning medium. The order parameters of two rigid hexosamine rings of I were determined separately using singular value decomposition and ab initio structures of disaccharide fragments of I. The order parameters were very similar implying that a common order tensor can be used to analyze the structure of I. Using one order tensor, RDCs of both hexosamine rings were used as restraints in molecular dynamics simulations. RDCs of the inner iduronic acid were calculated for every point of the molecular dynamics trajectory. The fitting of the calculated RDCs of the two forms of the iduronic acid to the experimental values yielded a population of C-1(4) and S-2(o) conformers of iduronic acid that agreed well with the analysis based on proton-proton scalar coupling constants. The glycosidic linkage torsion angles in RDC-restrained molecular dynamics (MD) structures of I are consistent with the interglycosidic three-bond proton-carbon coupling constants. These structures also show that the shape of heparin is not affected dramatically by the conformational flexibility of the iduronic acid ring. This is in line with conclusions of previous studies based on MD simulations and the analysis of H-1-H-1 NOEs. Our work therefore demonstrates the effectiveness of RDCs in the conformational analysis of glycosaminoglycans.