Mechanism of reductive protein unfolding.

The reductive unfolding of ribonuclease A with dithiothreitol proceeds through parallel pathways with the formation of two well-populated partially-unfolded three-disulphide intermediates. Two distinct local unfolding events rather than a global one are involved in the rate-limiting steps. These results are contrary to the current view that protein unfolding generally follows an all-or-none mechanism, and that the rate-limiting step is controlled by an extensive rearrangement of the native structure. Sequential breakage of disulphide bonds through local unfolding events is energetically more favourable than disruption of the native structure through global unfolding. The results also indicate that the oxidative refolding of ribonuclease A from the fully-reduced form proceeds through parallel conformationally-distinct transition states.