Heat-Triggered Conversion of Protofibrils into Mature Amyloid Fibrils of β2-Microglobulin

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Abstract
Heat-triggered conversion of the salt-induced thin and flexible protofibrils into well-organized thick and straight mature amyloid fibrils was achieved with β2-microglobulin, a protein responsible for dialysis-related amyloidosis. First, protofibrils that formed spontaneously at pH 2.5 in the presence of 0.5 M NaCl were aggregated by agitating the solution. Second, the aggregated protofibrils were heated in a cell of a differential scanning calorimeter (DSC). The DSC thermogram showed an exothermic transition with sigmoidal temperature dependence, resulting in a remarkably large decrease in the heat capacity of the solution. Third, on the basis of electron microscopy together with circular dichroism spectroscopy, seeding experiments, and a thioflavin T binding assay, the sigmoidal transition was found to represent the conversion of protofibrils into mature amyloid fibrils. Furthermore, DSC thermograms obtained at various heating rates revealed that the transition curve depends on the heating rate, implying that the effects of heat associated with the conversion to the mature fibrils are kinetically controlled, precluding an interpretation in terms of equilibrium thermodynamics. Taken together, these results highlight the importance of the change in heat capacity in addressing the biological significance of interactions between solvent water and amyloid fibrils and, moreover, in detecting the formation of amyloid fibrils.