Quaternary conformational stability: the effect of reversible self-association on the fibrillation of two insulin analogs.

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far-UV CD) and second derivative Fourier transform infrared (2D-FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α-helical in native insulin to predominantly β-sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self-associate. Composition gradient multi-angle light scattering (CG-MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant-induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first-order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers.