Rapid self-association of highly amyloidogenic H-fragments of insulin: Experiment and molecular dynamics simulations

The so-called ‘H-fragment’ of insulin is an extremely amyloidogenic double chain peptide consisting of the Nterminal parts of A-chain and B-chain linked by a disulfide bond between Cys-7A and Cys-7B. Here, we conduct a detailed investigation of the self-association behavior of H-fragment monomers into amyloid-like fibrils using kinetic assays, infrared spectroscopy, circular dichroism(CD), atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Unlike the intact predominantlyα-helical insulin, H-fragment remains in a disordered state in aqueous solutions. Its aggregation accelerateswith acidification of the environment leading, at pH 1.9, to the formation of thin and structurally homogenous fibrils with the infrared features typical for parallel β-sheet conformation. According to time-lapse AFM morphological analysis both secondary nucleation and fragmentation are involved in later stages of H-fibrils' self-assembly. Based on the low nucleation order (two) obtained fromthe global fitting of kinetic data, realistic all-atomMDsimulations of pairs of interacting H-fragmentmonomers were subsequently carried out. The molecular self-association scenario emerging from these simulations implicates the intrinsic conformational instability of H-monomer in its tendency to aggregate and formintermolecular β-sheet structure. Our findings provide the newmechanistic context for studies of insulin misfolding and aggregation