Mechanism of folding of ribonuclease A. Slow refolding is a sequential reaction via structural intermediates

Two models have been proposed to explain the observed folding kinetics of small proteins. The sequential model assumes that folding proceeds on an ordered pathway via structural folding intermediates, whereas the simple model of folding involves only multiple unfolded forms of the protein and a single native state. In the latter model, refolding is limited by interconversion reactions in the unfolded protein; accumulation of structural intermediates during folding is excluded. Here, two experimental tests are presented to discriminate between these models for the major slow folding species of ribonuclease A. The first test shows that a nativelike intermediate accumulates during folding, which unfolds rapidly compared to native ribonuclease A, and the second test demonstrates that refolding is a sequential reaction, resulting in the transient accumulation of an intermediate and in a lag in the formation of fully native protein. Both results rule out the simple model of folding and agree with the sequential model via structural intermediates. The nativelike intermediate is stable toward unfolding and is on the pathway of refolding for denaturant concentrations up to 2 M guanidine hydrochloride at pH 6 and 10 degrees C.