Elucidation of the disulfide-folding pathway of hirudin by a topology-based approach.

A theoretical model for the folding of proteins containing disulfide bonds is introduced. The model exploits the knowledge of the native state to favor the progressive establishment of native interactions. At variance with traditional approaches based on native topology, not all native bonds are treated in the same way; in particular, a suitable energy term is introduced to account for the special strength of disulfide bonds, as well as their ability to undergo intramolecular reshuffling. The model thus possesses the minimal ingredients necessary to investigate the much debated issue of whether the refolding process occurs through partially structured intermediates with native or non-native disulfide bonds. This strategy is applied to a context of particular interest, the refolding process of hirudin, a thrombin-specific protease inhibitor, for which conflicting folding pathways have been proposed. We show that the only two parameters in the model (temperature and disulfide strength) can be tuned to reproduce well a set of experimental transitions between species with different number of formed disulfides. This model is then used to provide a characterization of the folding process and a detailed description of the species involved in the rate-limiting step of hirudin refolding.