Carbon-13 chemical shifts on oxytocin as a consequence of its interaction with neurophysins

Carbon-13 NMR was used to study the interaction of the hormone oxytocin with neurophysin (NP). Oxytocins specifically enriched to 90% 13C in the alpha-carbons of Leu-3 (in [3-leucine]oxytocin), Gln-4, and Leu-8 and in the carbonyl carbon of Cys-6 were synthesized, so that the effect on these positions of binding to NP could be monitored. The alpha-carbons of residues 3 and 4 experienced shifts of -4.2 and -1.5 ppm (negative shifts are downfield), respectively, upon binding of the hormone to NP. The carbonyl carbon of residue 6 underwent a shift of +0.7 ppm, while the alpha-carbon of residue 8 displayed no shift. For each enriched residue, the hormone diastereoisomer in which this residue had the D configuration was also synthesized. NMR was then used to determine the binding affinity of the various diastereoisomers to NP, as well as to measure the NMR parameters of the bound peptides. When position 3 had the D configuration, the binding affinity for NP was 10-20% that of the native hormone. For positions 4, 6, and 8, the D diastereoisomers bound with the same affinity as oxytocin. The alpha-carbons of D residues of positions 3 and 4 shifted by -2.5 and +0.4 ppm, respectively, the carbonyl carbon of D-Cys-6 shifted by +1.4 ppm, and the alpha-carbon of D-Leu-8 was unshifted on binding to NP. The shift and diastereoisomer binding data, combined with previous results involving enriched carbons and/or diastereoisomers of residues 1, 2, and 9, support the conclusion that residues 1 and 2 are most crucial for binding of oxytocin to NP, residue 3 is less important, and residues 4-9 are of only slight significance.(ABSTRACT TRUNCATED AT 250 WORDS)