A VERY FAST PHASE IN THE REFOLDING OF DISULFIDE-INTACT RIBONUCLEASE-A - IMPLICATIONS FOR THE REFOLDING AND UNFOLDING PATHWAYS

The refolding and unfolding of disulfide-intact ribonuclease A has been studied by using single-jump and double-jump stopped-flow techniques. Absorbance and fluorescence detection methods were used to follow the kinetics. By appropriate choice of solution conditions (1.5 M guanidine hydrochloride, pH 3.0, at temperatures less than or equal to 15 degrees C) to slow the refolding process, a new very fast phase has been observed in addition to the usual fast and slow phases that involve the unfolded species U-f and U-S, respectively. Double-jump experiments consisting of an unfolding step at 4.2 M guanidine hydrochloride and pH 2.0 followed by a refolding step at 1.5 M guanidine hydrochloride and pH 3.0 were carried out to monitor the unfolding process. These experiments demonstrated that the new phase arises from a separate unfolded species, U-vf, which is present to the extent of about 6% in the equilibrium ensemble of unfolded protein at high guanidine hydrochloride concentration and low pH. A new model for the unfolding pathway and interconversion among unfolded species is proposed based on two independent isomerization processes. The equilibrium constants and activation energies obtained for each process suggest that they involve the isomerization of cis prolines. We propose that the isomerizations occur at the X-Pro peptide bonds of Pro 93 and 114. In the model, U-vf is the first species to form without isomerization at any cis X-Pro peptide bonds when the native protein is unfolded; U-f and U-S then form from U-vf through two independent isomerization processes. Both prolines are in the native (cis) conformation in U-vf. In U-f, Pro 114 is in a nonnative (trans) conformation while, in U-S, Pro 93 is in a nonnative (trans) conformation. The slow folding species, U-S, actually consists of (at least) two species: U-S(alpha), with Pro 93 in a nonnative (trans) conformation and U-S(beta), with both Pro 93 and 114 in nonnative (trans) conformations. Finally, the kinetic data suggest that the presence of a nonnative trans conformation at the Tyr 92-Pro 93 peptide bond impedes the refolding rate of ribonuclease A much more than the presence of a nonnative trans conformation at the Asn 113-Pro 114 peptide bond.