Carbon-13 NMR was used to study the interaction of the hormone oxytocin with neurophysin (NP). Oxytocins specifically enriched to 90% 13C in the α-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 α-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 α-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 α-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 α-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. An unequivocal interpretation of the chemical shift effects was not possible, but side chain conformation was deduced to contribute significantly. It appears that the conformations of several side chains of oxytocin bound to NP differ significantly from the predominantly averaged side chain conformations present in solution. For Cys-1, Tyr-2, and Leu-3, the change in side chain conformation is caused by direct interaction of the side chain with NP. For Gln-4, the side chain does not directly interact with NP, but its bound conformation differs from its free conformation due to changed interactions with the rest of the oxytocin molecule. The overall dissociation rate constant of oxytocin from NP was found to be 2-4 s-1, in good agreement with results from rapid kinetic experiments. Substitution of a D residue in position 3, which resulted in a decreased binding affinity for NP, also led to an increased dissociation rate constant, while the association rate constant for the D diastereoisomer was the same or somewhat higher than that of the unmodified peptide. Thus, the weaker binding of the D diastereoisomer is due to a weaker complex rather than to an unfavorable conformation of the free hormone.
|Original language||English (US)|
|Number of pages||9|
|Publication status||Published - 1984|
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