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

Abstract

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.

Selective and stoichiometric incorporation of ATP by self-assembling amyloid fibrils.

“…1a) but reveals its extremely amyloidogenic character when separated from it. 34,35 When attached to certain moderately long non-amyloidogenic peptide chains ACC1-13 enforces cooperative amyloidogenic self-assembly and adoption of the common parallel β-sheet structure. 36 In this study, the amino acid sequence of ACC1-13 was extended at its C-end by an octalysine (K8) segment (Fig.…”

Multiscale Modeling of Amyloid Fibrils Formed by Aggregating Peptides Derived from the Amyloidogenic Fragment of the A-Chain of Insulin

“…The purity of the peptide was assessed by the supplier using MS-UPLC analysis (mass spectrometry coupled to ultra-performance liquid chromatography) and exceeded 96%. The stock freeze-dried peptide was solubilized according to the previously described protocol [15], consisting of dispersion of solid pellets in 8M guanidine hydrochloride (GdnHCl) solution, pH 9.0, at a 6 mg/mL peptide concentration followed by 30 min incubation at room temperature. The clear peptide solution was centrifuged at 13,400 rpm for 5 min to remove traces of insoluble matter.…”

“…Certain aspects of the predicted structures of ACC 1-13 fibrils, such as β-sheet content, diameter, and periodicity of the twisted surface (helical pitch), can, in principle, be compared with easily accessible experimental data. Hence, we have carried out de novo fibrillization of ACC 1-13 according to the established protocol [15]. The kinetic trajectories reflecting the rapid growth of amyloid mass shown in Figure 6A were obtained using fluorescence of Thioflavin T (ThT), an amyloid-specific molecular rotor whose quantum yield of fluorescence increases by 2-3 orders of magnitude upon intercalation onto the amyloid surface [45].…”

“…As a model for our CABS-dock docking simulations, peptide ACC 1-13 -an extremely amyloidogenic N-terminal fragment of the insulin A-chain-was selected [15][16][17]. The ACC 1-13 peptide comprises the first 13 residues of the A-chain and retains the intrachain Cys6-Cys11 disulfide bond, while the Cys7 residue, involved in the interchain disulfide bond in the parent insulin monomer, is replaced with Ala. We have shown earlier that ACC 1-13 is the key amyloidogenic region of the larger highly aggregation-prone H-fragment, which is released upon partial digestion of insulin with pepsin [18].…”

“…The ACC 1-13 peptide comprises the first 13 residues of the A-chain and retains the intrachain Cys6-Cys11 disulfide bond, while the Cys7 residue, involved in the interchain disulfide bond in the parent insulin monomer, is replaced with Ala. We have shown earlier that ACC 1-13 is the key amyloidogenic region of the larger highly aggregation-prone H-fragment, which is released upon partial digestion of insulin with pepsin [18]. The intact disulfide bond is not only compatible with the formation of a β-sheet structure but also accelerates the process, possibly through the entropic destabilization of free disordered monomers [15]. The disordered state of ACC 1-13 before the formation of fibrils appears to contribute to the overall aggregation rate by minimizing energy barriers and kinetic traps.…”