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

TL;DR

This paper introduces a topology-based theoretical model to study protein disulfide bond folding, applied specifically to Hirudin, revealing insights into its folding pathway and the role of native and non-native disulfide bonds.

Contribution

A novel model that incorporates disulfide bond strength and reshuffling, providing new understanding of the folding pathway of Hirudin with minimal parameters.

Findings

Model accurately reproduces experimental folding transitions.

Identifies key species involved in the rate-limiting step.

Highlights the importance of disulfide bond reshuffling in folding.

Abstract

A theoretical model for the folding of proteins containing disulfide bonds is introduced. The model exploits the knowledge of the native state to favour 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 (irrespective of whether they are native or not) as well as their ability to undergo intra-molecular reshuffling. The model thus possesses the minimal ingredients necessary to investigated the much debated issue of whether the re-folding process occurs through partially structured intermediates with native or non-native disulfide bonds. This strategy is applied to a context of particular interest, the re-folding 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 disulfide. This model is then used to provide a characterisation of the folding process and a detailed description of the species involved in the rate-limiting step of Hirudin refolding.