Although amyloid fibrils are associated with a number of serious disorders, the mechanism of amyloid fibrillation remains elusive. Because many structurally unrelated proteins can form amyloid fibrils, amyloid misfolding is considered a general property of polypeptide chains. Previously, to visualize amyloid fibrils, we combined total internal reflection fluorescence microscopy (TIRFM) with amyloid-specific thioflavin T (ThT) fluorescence1 . With this approach, we succeeded in observing the growth of amyloid fibrils in real-time at a single fibrillar level.
Here, we show that amyloid fibrillation is determined by equilibrium solubility of unfolded proteins and that supersaturation prevents conformational transition2 . Under the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered fibrillation3 4 . The optimal alcohol concentration depended on the alcohol species and was opposite to the equilibrium solubility profiles of lysozyme in water/alcohol mixtures. The results indicate that amyloid fibrillation is a combined effect of decreased solubility and supersaturation and that ultrasonication is effective in breaking supersaturation and thus producing amyloid fibrils..
With β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetery (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils. The results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.