Expression, folding, and thermodynamic properties of the bovine oxytocin-neurophysin precursor: Relationships to the intermolecular oxytocin-neurophysin complex

Earlier thermodynamic studies of the intermolecular interactions between ma ture oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address thi s issue, the disulfide-rich precursor of oxytocin-associated bovine neuroph ysin was expressed in Escherichia coli and folded in vitro to yield milligr am quantities of purified protein; evidence of significant impediments to y ield resulting from damage to Cys residues is presented. The inefficiency a ssociated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent wit h this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding be tween oxytocin and neurophysin in the precursor had only a small thermodyna mic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unligan ded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of stru ctural differences between the precursor and the intermolecular complex, wh ich persist beyond the pH 10 transition, suggests that the covalent attachm ent of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are dis tinct from those of either the liganded or unliganded mature protein.