THERMODYNAMIC ROLE OF THE PRO REGION OF THE NEUROPHYSIN PRECURSOR IN NEUROPHYSIN FOLDING - EVIDENCE FROM THE EFFECTS OF LIGAND PEPTIDES ON FOLDING

Attention has focused recently on the role of amino-terminal precursor pro regions in protein folding, with particular emphasis on their eff ects on folding kinetics. We examined the kinetic and thermodynamic ef fects of ligand peptides on the folding of neurophysin from the reduce d state; these peptides serve as analogs of the pro regions of the com mon precursors of the neurophysins and the hormones oxytocin and vasop ressin. Folding of reduced, mononitrated bovine neurophysin-II was mon itored by circular dichroism in a glutathione redox buffer. The result s confirmed the ability of neurophysin to fold to a limited extent (20 -25% in this system) in the absence of ligand peptides, Ligand peptide s increased the efficiency of folding to 100%, the exact efficiency be ing dependent on peptide identity and concentration. However, the rate of folding was peptide-independent, Analysis of the folding reaction demonstrated relatively rapid conversion of the reduced state to a dis ulfide-scrambled state, which slowly converted (half-life of 5 h at pH 7.3) to the folded state, Native unliganded neurophysin also equilibr ated with the disulfide-scrambled state in the same redox buffers. For each peptide, an equilibrium constant for the folding reaction, repre senting the amount of peptide bound in the folding system as a functio n of peptide concentration, was calculated. Comparison of this constan t with the intrinsic binding constants of the native protein allowed t he derivation, under conditions at or approaching thermodynamic revers ibility, of the relative stability of the native and disulfide-scrambl ed states. The results indicate that the scrambled state, which probab ly represents the presence of incorrect disulfide pairs in both protei n domains, is more stable than the native unliganded state by similar to 1 kcal/mol in this system. The role of ligand peptide therefore is to stabilize the folded protein after it is formed, i.e., it provides a thermodynamic sink, The results contrast with the putative behavior of exogenous peptides representative of the pro regions of subtilisin and alpha-lytic protease, which are generally considered to facilitate folding by reaction with folding intermediates. A potential alternati ve view of the role of propeptides in protease folding is suggested.