IDENTIFICATION OF THE UNSTABLE NEUROPHYSIN DISULFIDE AND LOCALIZATIONTO THE HORMONE-BINDING SITE - RELATIONSHIP TO FOLDING-UNFOLDING PATHWAYS

For unliganded neurophysin, the effects of reduction of a single disul fide and limited regeneration of activity following reduction have sug gested metastable disulfide pairing relative to that of the neurophysi n 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 mimickin g precursor folding. To assign the source of the metastability of the unliganded mature protein, the disulfide(s) most susceptible to reduct ion and the last to be reoxidized following complete reduction were id entified. Partial reduction of the first disulfide followed by trappin g of the generated thiols with [C-14]iodoacetate gave a distribution o f label consistent with identification of the unstable disulfide as th e 10-54 bridge and rapid interchange of the Cys-10 thiol with other di sulfides in the amino-terminal disulfide domain. The same thiol distri bution was seen at the terminal stage of reoxidation following complet e reduction, providing evidence that unfolding and folding pathways ar e 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 th e 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 ami no domain are homologous to bridges in the carboxyl-terminal domain th at do not appear to be unstable, the results allow the possibility tha t the 10-54 bridge, which is unique to the amino domain, destabilizes other disulfides in that domain.