Folding Kinetics of Two-state Proteins: Effect of Circularization, Permutation, and Crosslinks

Protein folding kinetics has recently been probed by clever experiments using circular permutants and other topological mutations. A circular permutant is created from a wild-type protein by covalently linking together the chain ends and cleaving elsewhere in the chain. An interesting puzzle is why circular permutation causes no apparent change in the folding mechanism of CI2, but dramatic changes in the folding mechanisms of S6 and of an SH3 domain, as determined by Φ-value experiments. Here, we use a computational model to predict the folding routes of topological variants, based on a measure (effective contact order) of the chain entropy loss at each folding step. The predictions are consistent with the experiments, leading to insights into the folding routes and into the meaning of Φ-values in general. We find that Φ-values do not always describe time sequences of folding events, or positions along a single reaction coordinate; rather, Φ reflects only the degree of rate control. For example, the circular permutant P40−41 of CI2 is predicted to reverse the time sequence of the formation of β1β4 relative to β2β3, without changing the diffuse Φ-value distribution, while the circular permutant P13−14 of S6 switches the rate-limiting step from the formation of β1β4 to β1β3, changing the Φ-value distribution from diffuse to strongly polarized. As a test of the model, we propose mutations that should reverse these outcomes.