Modulation of allosteric interactions in neurophysin induced by succinylation of serine-56 or cleavage of residues 1-8.
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)
Huang
H
H
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
LaBorde
T
T
Breslow
E
E
D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008969:Molecular Sequence Data; D009481:Neurophysins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D012694:Serine; D013387:Succinic Anhydrides; D013912:Threonine
D009481:Neurophysins; D010446:Peptide Fragments; D013387:Succinic Anhydrides; D013912:Threonine; D012694:Serine; C031801:succinic anhydride