Effect of Co-Solutes On the Aggregation of the Model Amyloid Protein Insulin

In vitro experiments have been widely used to characterize the misfolding/unfolding pathway characteristic of amylodogenic proteins.  Conversion from natively folded amyloidogenic proteins to oligomers via nucleation is the accepted path to fibril formation.  In order to simulate the crowded environment of cells (protein concentration ~ 200 mg/ml), we investigated the aggregation kinetics of insulin, a well-established amyloid model protein, in the presence of different co-solutes including:  macromolecules, such as polyvinylpyrrolidone (PVP), MW 40 kDa, or micromolecules such as glycerol (Gly), MW 92 Da.  Using absorbance spectroscopy to measure turbidity of the solution, the rate of aggregation was measured to determine the effects co-solutes on insulin aggregation kinetics.  AFM imaging was used to investigate the morphology of the aggregates and rheometry to measure the solution viscosity for different concentrations of co-solutes.  The results show that (a) addition of co-solutes substantially delayed formation of fibrils, and (b) the delay was more pronounced for PVP than Gly.  The results were confirmed using fluorescence correlation spectroscopy.  Either viscosity and reduced solute diffusion or specific interaction effects of the co-solutes on the reaction rates of oligomers prior to the formation of fibrils are used to explain these results.