Modulation of allosteric interactions in neurophysin induced by succinylation of serine-56 or cleavage of residues 1-8

Abstract

Neurophysin is an allosteric protein in which peptide binding and self-association are positively linked. Reaction of neurophysin with succinic anhydride led to a large decrease in peptide affinity assignable to succinylation of a serine or threonine hydroxyl group. To identify the residue involved, acetimidated protein was reacted with [14C]succinic anhydride and the active and inactive components were separated by affinity chromatography. Performic acid oxidation and tryptic and Asp-N mapping of the two components, followed by automated Edman degradation, allowed identification of the critical residue as Ser-56. This residue is not a direct participant in peptide binding and is distant from the subunit interface of the dimer, but it is immediately adjacent to the site of one of the known mutations associated with familial diabetes insipidus. Examination in solution of the peptide affinity of neurophysin succinylated at Ser-56 indicated a binding affinity approximately 1/20th that of the native protein or of protein succinylated at other residues, and a loss of the normal dependence of binding affinity on protein concentration. Under the same buffer conditions, loss of the concentration dependence of binding, in addition to the previously demonstrated loss of binding affinity, also accompanied excision of residues 1-8, an effect attributed to the loss of binding site residue Arg-8. However, in contrast to the effects of succinylation on native neurophysin, only minor effects of succinylation on the binding affinity of the des-1-8 protein were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

“…This modification was shown previously to lead to a large decrease in binding constant (25). Table II demonstrates a reduction in dimerization in the unliganded state by a factor of 2-3 when NP succinylated at Ser-56 is compared with protein similarly modified with the exception of Ser-56 (see "Materials and Methods").…”

“…This is similarly suggested by the effects on dimerization of succinylation of Ser-56. However, mutation of Gly-57 has so far not reduced ligand-facilitated dimerization, whereas succinylation of Ser-56 appears to block this process (25), suggesting that the Ser-56 modification has a greater effect than the Gly-57 mutations on dimerization in the liganded state.…”

“…Chemical and Enzymatic Modification of Wild Type Protein-Succinylated BNP-I was prepared from native BNP-I as described elsewhere (25). Following succinylation, protein retarded by the affinity column is succinylated at its two Lys residues and randomly succinylated at Ser residues exclusive of Ser-56, whereas protein not retarded by the affinity column differs from the retarded fraction by its succinylation at Ser-56 (25).…”

“…Ref. 25). Protein modeling utilized the Swiss Protein Data Bank modeling system and the crystal structures of des-1-6 bovine NP-II in its unliganded state 2 and as its lysine vasopressin complex.…”

Expression, Folding, and Thermodynamic Properties of the Bovine Oxytocin−Neurophysin Precursor: Relationships to the Intermolecular Oxytocin−Neurophysin Complex

“…The fraction of correctly folded protein, f, is calculated from the observed ratio, R, of optical activity at 240 nm to that at 290 nm from the relationship Denaturation Studies. The stabilities to guanidine denaturation of the folded precursor and of bovine NP-I, the latter prepared and affinity-purified as described previously (19), were compared. Solutions of protein in 0.1 M ammonium acetate and different concentrations of freshly dissolved guanidine hydrochloride (Sigma, 99% grade) were prepared from a common stock protein solution, and the CD spectrum of each was obtained in the region 340-240 nm.…”