Beyond amino acid sequence: disulfide bonds and the origins of the extreme amyloidogenic properties of insulin's H-fragment.

The presence of disulfide bonds affects the protein stability and therefore tendency to misfold and form amyloid-like fibrils. Insulin's three disulfide bridges stabilize the native state and prevent aggregation. Partial proteolysis of insulin releases highly amyloidogenic and inherently disordered two-chain 'H-fragment' retaining insulin's Cys7A-Cys7B and Cys6A-Cys11A disulfide bonds. The abrupt self-association of H-fragment monomers into fibrils is suppressed in the presence of disulfide-reducing agent. These circumstances make the H-fragment an interesting model to study the impact of disulfide bonds on amyloidogenesis beyond the 'stabilization-of-the-native-state' paradigm. Here, we investigate fibrillization of various synthetic peptides derived from the H-fragment through modifications of Cys7A-Cys7B/Cys6A-Cys11A bonds. In comparison to H-fragment, aggregation of a two-chain 'AB' analog lacking Cys6A-Cys11A bond is decelerated, while the alternative removal of Cys7A-Cys7B bond releases a non-aggregating B-chain and a highly amyloidogenic 'ACC' fragment containing the intrachain Cys6A-Cys11A bond. Our analysis, supported by calculations of configurational entropy, suggests that Cys6A-Cys11A bond is a key factor behind the explosive self-association of H-fragment. The bond restricts the conformational space probed by nucleating monomers which is reflected by an approximately 2.4 kJ·mol-1 K-1 decrease in entropy. The fact that the intact Cys6A-Cys11A bond promotes fibrillization of the H-fragment is remarkable in light of the previously established role of the same disulfide bond in preventing formation of insulin fibrils. Our results imply that a single disulfide bond within a folded protein and its fragment may play entirely different roles in aggregation and that this role may evolve with progressing phases of misfolding.