| Thermodynamics and Kinetics of Insulin Fibril Formation The deposition of amyloid, insoluble fibrillar aggregate composed of normally soluble cellular proteins, is a major symptom of diseases such as Alzheimer's, type II diabetes and the spongiform encephalopathies. The deposits are believed to play a central role in the pathogenesis of these diseases but the mechanism by which normally soluble proteins are transformed into fibrillar aggregates is poorly understood. The hormone insulin has been shown to form similar fibrils on heating in acidic solution, and has been used previously as a model for amyloid. With this in mind, the formation of insulin fibrils in solution, as a model for amyloid fibril formation, was studied using a combination of calorimetric and kinetic techniques. At pH 2.0, the formation of fibrils from native bovine insulin is exothermic and shows a significant decrease in excess heat capacity. Pressure perturbation calorimetry (PPC) shows that the fibrils have a lower thermal expansion coefficient (alpha) than the native protein under the same conditions. This contrasts with the increase in heat capacity and expansion coefficient observed with thermally misfolded protein and non-fibrillar aggregates of control proteins. The kinetics of fibril formation, followed by differential scanning and isothermal calorimetry together with turbidometric and thioflavin T fluorescence methods, show the classic lag phase associated with nucleation-growth mechanisms. This is supported by seeding experiments involving the addition of pre-formed fibrils, which reduce the lag phase. Cyclodextrins inhibit the kinetics of fibrillation in a manner consistent with the anticipated interaction of these cyclic polysaccharides with hydrophobic groups on the insulin molecule. Overall, the thermodynamic data indicate that the molecular packing of polypeptides in insulin fibrils formed under these conditions is at least as dense as in the native fold. |
