The effects of various membrane physical-chemical properties on the aggregation kinetics of insulin.

In a simplified approach to the in vivo situation, where pathogenic fibrillar protein deposits are often found associated with cellular membranes, the aggregation kinetics of insulin in the presence of various model biomembranes were investigated using the Thioflavin T (ThT) fluorescence assay. The lipid dynamics near the gel-fluid transition, the chain length of saturated lipids and the presence of DOPE or DOPS in DOPC-vesicles modulate the aggregation kinetics of insulin in an indifferent, an aggregation-accelerating or an aggregation-inhibiting manner, subtly depending on the pH-value and the presence of salt. The rate of insulin aggregation in bulk solution dominates the overall aggregation process in most cases at low pH, where the lipid additives exert no effect on the aggregation kinetics.

Cytotoxicity of insulin within its self-assembly and amyloidogenic pathways.

Solvational perturbations were employed to selectively tune the aggregational preferences of insulin at 60 degrees C in vitro in purely aqueous acidic solution and in the presence of the model co-solvent ethanol (EtOH) (at 40%(w/w)). Dynamic light scattering (DLS), thioflavin T (ThT)-fluorescence, Fourier transform infrared (FTIR) and atomic force microscopy (AFM) techniques were employed to characterize these pathways biophysically with respect to the pre-aggregational assembly of the protein, the aggregation kinetics, and finally the aggregate secondary structure and morphology. Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the insulin species that form in the two distinct aggregation pathways.

Solvation-assisted pressure tuning of insulin fibrillation: from novel aggregation pathways to biotechnological applications.

Solvation-assisted pressure tuning has been employed to unravel unknown structural and kinetic aspects of the insulin aggregation and fibrillation process. Our approach, using fluorescence, Fourier transform infrared and atomic force microscopy techniques in combination with pressure and solvent perturbation, reveals new insights into the pre-aggregated regime as well as mechanistic details about two concurrent aggregation pathways and the differential stability of insulin aggregates. Pressure uniformly fosters the dissociation of native insulin oligomers, whereas the aggregation pathways at elevated temperatures are affected by pressure differently and in a cosolvent-dependent manner.

Solvational tuning of the unfolding, aggregation and amyloidogenesis of insulin.

Solvational perturbations, accomplished by the addition of the three model cosolvents glycerol, ethanol and trifluoroethanol, exert pronounced and diversified effects on the unfolding, non-native assembly and fibril formation of the amyloidogenic protein insulin. Fluorescence, CD and UV-spectroscopic methods as well as atomic force microscopy imaging have been employed to reveal distinct structural and kinetic features upon the aggregation of insulin under different solvational perturbations, which ultimately manifest in morphological variations of mature aggregates and fibrils. In particular, fluorescence anisotropy studies proved to be very valuable in characterizing the corresponding aggregation nuclei.

Ethanol-perturbed amyloidogenic self-assembly of insulin: looking for origins of amyloid strains.

A model cosolvent, ethanol, has profound and diversified effects on the amyloidogenic self-assembly of insulin, yielding spectroscopically and morphologically distinguishable forms of beta-aggregates. The alcohol reduces hydrodynamic radii of insulin molecules, decreases enthalpic costs associated with aggregation-prone intermediate states, and accelerates the aggregation itself. Increasing the concentration of the cosolvent promotes curved, amorphous, and finally donut-shaped forms.

High pressure promotes circularly shaped insulin amyloid.

Amyloids, initially associated with certain degenerative diseases, and recently with the prions and prion-based inheritance in yeasts, are linearly-ordered beta-sheet-rich protein aggregates, presently thought to represent a rather common generic trait of proteins as polymers. Regardless of genetic origins and properties of precursor protein molecules, amyloids share many physicochemical properties, including the linear fibrillar morphology. Here, we show that under high hydrostatic pressure insulin forms amyloids of a unique circular morphology.