Exothermic Effects Observed upon Heating of β2-Microglobulin Monomers in the Presence of Amyloid Seeds

To understand the initial stages in the formation of amyloid fibrils of β2-microglobulin, a protein responsible for dialysis-related amyloidosis, the effects of heat on the acid-unfolded monomer at pH 2.5 were studied. In the presence of a low concentration of seed fibrils, differential scanning calorimetric thermograms of acid-unfolded β2-microglobulin monomers showed a large decrease in heat capacity with a sigmoidal temperature-dependence, which was subsequently released at higher temperature. Measurements of circular dichroism, atomic force microscopy, ultracentrifugation, and repeated differential scanning calorimetry indicated that the exothermic sigmoidal transition is accompanied by the conversion of about 12% of the monomeric β2-microglobulin molecules into amyloid fibrils, which subsequently dissociate into monomers at high temperature. Interestingly, amyloid fibrils, formed partly after the sigmoidal transition, exhibited a heating rate-dependent, kinetically controlled thermal response, indicating that 12% of the total protein is enough to exhibit the unique thermal response. On the other hand, the salt-induced protofibrils did not show such a calorimetric response, indicating that the kinetic thermal response is unique to the particular structure of fibrils. Taken together, although the calorimetric behavior of amyloid fibrils remains elusive, it may be interpreted in terms of the effects of heat associated with the formation, the association, and the unfolding of fibrils, in which the interactions between specific β-sheet structures and water molecules play a crucial role and are sensitively reflected in the heat capacity change in protein solution.