title: Quaternary conformational stability: the effect of reversible self-association on the fibrillation of two insulin analogs.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Center for Pharmaceutical Biotechnology, University of Colorado, Boulder, Colorado 80309; telephone: +1-303-492-4776; fax: +1-303-492-4341.
authors.forename: D Brett
authors.initials: DB
authors.lastname: Ludwig
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Webb
authors.forename: Cristina
authors.initials: C
authors.lastname: Fernández
authors.forename: John F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: Theodore W
authors.initials: TW
authors.lastname: Randolph
mesh_terms: D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry*; D066329:Protein Aggregates; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D007328:Insulin; D066329:Protein Aggregates