Expression, Folding, and Thermodynamic Properties of the Bovine Oxytocin-Neurophysin Precursor: Relationships to the Intermolecular Oxytocin-Neurophysin Complex † Sharon Eubanks, ‡ Min Lu, ‡ David Peyton, § and Esther Breslow* ,‡ Department of Biochemistry, The Joan and Sanford I. Weill Medical College of Cornell UniVersity, 1300 York AVenue, New York, New York 10021, and Department of Chemistry, Portland State UniVersity, Portland, Oregon 97207-0751 ReceiVed June 8, 1999; ReVised Manuscript ReceiVed August 16, 1999 ABSTRACT: Earlier thermodynamic studies of the intermolecular interactions between mature 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 this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with 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 between oxytocin and neurophysin in the precursor had only a small thermodynamic 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 unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein. The hormones oxytocin and vasopressin are each synthe- sized as part of a larger precursor (1, 2) that also contains the protein neurophysin (NP). 1 The covalent linkage between hormone and NP segments is cleaved by processing, yielding mature hormone and NP which remain noncovalently bonded within neurosecretory granules prior to release into the blood (3). The neurophysins associated with each hormone are nonidentical, but are structurally homologous with very similar properties in vitro (3, 4). The noncovalent interactions between NP, oxytocin, and other ligand peptides have been extensively studied in solution (3), and crystal structures for the complexes of bovine NP-II (vasopressin-associated) with a ligand dipeptide (5) and with oxytocin (6) have been determined. However, relatively little is known about precursor properties. Such properties are of particular interest in view of the importance of the precursor to the proper targeting of hormone to regulated neurosecretory granules and the failure of such targeting in diabetes insipidus, a disorder characterized by insufficient vasopressin production (7, 8). Chaiken and co- workers (9, 10) prepared the bovine oxytocin precursor by semisynthesis, chemically ligating the hormone-containing segment to biologically derived bovine NP-I. A central conclusion of their work was that interactions between hormone and NP segments within the precursor were analogous to those within processed hormone-NP com- plexes, and conferred on the precursor self-association properties analogous to those of the complexes. The precursor was also demonstrated to be more stable than unliganded NP (9), but the additional stability was not quantitated. In this work, we report the first expression in Escherichia coli of the bovine oxytocin precursor, and investigation of its thermodynamic, structural, and folding properties. The thermodynamic and structural properties of the precursor were of interest in part because they provide the opportunity to compare intramolecular hormone-NP interaction with the intermolecular interaction between mature hormone and mature NP. The noncovalent interaction of NP with ligand † Supported by NIH Grant GM-17528 and NIH Fellowship F31 NS- 10204 to S.E. * To whom correspondence should be addressed: Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. Telephone: (212) 746-6428. Fax: (212) 746-329. E-mail: ebreslow@mail.med.cornell.edu. ‡ The Joan and Sanford I. Weill Medical College of Cornell University. § Portland State University. 1 Abbreviations: NP, neurophysin; bovine NP-I, bovine oxytocin- associated neurophysin; bovine NP-II, bovine vasopressin-associated neurophysin; CD, circular dichroism; GSH, reduced glutathione; GSSG, oxidized glutathione; SDS, sodium dodecyl sulfate; PAGE, polyacryl- amide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorp- tion ionization time-of-flight mass spectrometry; NMR, nuclear mag- netic resonance; TOCSY, two-dimensional total correlation J spectros- copy. 13530 Biochemistry 1999, 38, 13530-13541 10.1021/bi9912950 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/24/1999