Identification of the unstable neurophysin disulfide and localization to the hormone-binding site. Relationship to folding-unfolding pathways.
For unliganded neurophysin, the effects of reduction of a single disulfide and limited regeneration of activity following reduction have suggested metastable disulfide pairing relative to that of the neurophysin precursor. This metastability was confirmed in the present study by the demonstration of almost complete regeneration of activity from the reduced state in the presence of ligand peptides, conditions mimicking precursor folding. To assign the source of the metastability of the unliganded mature protein, the disulfide(s) most susceptible to reduction and the last to be reoxidized following complete reduction were identified. Partial reduction of the first disulfide followed by trapping of the generated thiols with [14C]iodoacetate gave a distribution of label consistent with identification of the unstable disulfide as the 10-54 bridge and rapid interchange of the Cys-10 thiol with other disulfides in the amino-terminal disulfide domain. The same thiol distribution was seen at the terminal stage of reoxidation following complete reduction, providing evidence that unfolding and folding pathways are the same at this stage. The results indicate that, in the absence of bound peptide, the state with correct pairing of the 10-54 bridge has no significant thermodynamic advantage over interchanged states of the amino-terminal domain. However, since the 10-54 bridge is located at the peptide-binding site, the correct pairing is directly stabilized by ligand peptides. Moreover, since the other three bridges of the amino domain are homologous to bridges in the carboxyl-terminal domain that do not appear to be unstable, the results allow the possibility that the 10-54 bridge, which is unique to the amino domain, destabilizes other disulfides in that domain.
Huang
H B
HB
Department of Biochemistry, Cornell University Medical College, New York, New York 10021.
Breslow
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism*; D006728:Hormones / Q000378:metabolism*; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009481:Neurophysins / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases; D013816:Thermodynamics
D004220:Disulfides; D006728:Hormones; D009481:Neurophysins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases