NMR studies of fibronectin peptides and their interactions with heparin and related glycosaminoglycans

{dollar}sp1{dollar}H and {dollar}sp{lcub}13{rcub}{dollar}C Nuclear Magnetic Resonance (NMR) spectroscopy at 8.5 T and circular dichroism (CD) were used to study the interactions of cell surface model compounds, heparin and chondroitin sulfate glycosaminoglycans (GAGs), with synthetic peptides, FN-C/H-I (YEKPGSPPREVVPRPRPGV) and FN-C/H-III (YRVRVTPKEKTGPMKE), from the 33 kD heparin binding domain of the extracellular matrix protein fibronectin.; Complete {dollar}sp1{dollar}H and {dollar}sp{lcub}13{rcub}{dollar}C NMR signal assignments for FN-C/H-III and partial assignments for FN-C/H-I were made using conventional one and two dimensional methods. FN-C/H-III has a +3 charge, while FN-C/H-I has a +2 charge at physiological pH. At pH 4.5 and in the presence of the highly anionic heparin biopolymer, these peptides formed insoluble complexes which redissolved completely at heparin carboxylate to peptide ratios of 3.5 for FN-C/H-III and 2.2 for FN-C/H-I.; Chemical shift, NOE, and relaxation data suggest that heparin interacts primarily with the two arginine residues located near the N-terminus of FN-C/H-III. Ionization of this peptide's two glutamic acid residues is suppressed in the presence of heparin, allowing adjacent lysine residues to become more favorable as secondary sites of interaction. NMR and CD data indicate that FN-C/H-III has a random secondary structure in solution and that heparin interactions do not cause a significant change in the overall conformation of the peptide. NOE data suggest that this peptide causes a rotation about the heparin I1 to A4 anomeric linkage.; For FN-C/H-I, NMR data imply that heparin interacts with multiple basic sites distributed throughout the fragment. NMR and CD data suggest that FN-C/H-I undergoes a greater conformational change upon association with heparin than does FN-C/H-III. NOE data do not clearly indicate a conformational change for heparin upon association with FN-C/H-I.; The addition of chondroitin sulfate GAGs to FN-C/H-III resulted in the formation of slowly exchanging complexes as evidenced by unique NMR signals which were most notable at GAG disaccharide to peptide ratios of 2.0 to 2.8. CD data indicate that chondroitin-6-sulfate and dermatan sulfate interact more strongly with FN-C/H-III than chondroitin-4-sulfate does. Each GAG appeared to affect the conformation of the peptide more significantly than heparin.