title: Modulation of dimerization, binding, stability, and folding by mutation of the neurophysin subunit interface.
abstract: Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.
authors.affiliation: Department of Biochemistry, The Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, New York, New York 10021, USA.
authors.forename: S
authors.initials: S
authors.lastname: Eubanks
authors.forename: T L
authors.initials: TL
authors.lastname: Nguyen
authors.forename: D
authors.initials: D
authors.lastname: Peyton
authors.forename: E
authors.initials: E
authors.lastname: Breslow
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009481:Neurophysins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010121:Oxytocin / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013552:Swine
chemical_list: D009481:Neurophysins; D006639:Histidine; D010121:Oxytocin; D001120:Arginine