Revisiting the mystery of temperature-induced degradation of amyloid seeds under dry conditions: A comparative study on two structural variants of insulin fibrils.

The remarkable thermodynamic stability of amyloid fibrils is paralleled by their heat-tolerance, which is also thought to underlie the resistance of prion infectivity to high temperatures. Here, we studied heat-stability of two structural variants of insulin fibrils: one formed in acidified aqueous solutions and the other formed in the presence of acetone. While heating under a dry N2 atmosphere leads to the release of water before the temperature reaches 120 °C, this process is not accompanied by changes in the secondary structure of fibrils, as judged by IR spectroscopy, or in their ability to seed native insulin. However, the seeding capacity is lost by both polymorphs upon exposure to 175 °C, even though, the corresponding heat effects are negligible. Interestingly, the 175 °C temperature treatment hardly affects the Raman spectral characteristics of either type of fibrils. According to liquid chromatography and mass spectrometry analyses of annealed fibrils dissolved in guanidine hydrochloride (GdnHCl), insulin with an intact covalent structure is prevalent in the soluble fraction. However, after heating at 175 °C, both variants lose the capacity to refold to the native insulin upon dilution of GdnHCl-treated samples, as monitored by far-UV circular dichroism (CD) spectra. We rationalize these findings by proposing that the high-temperature treatment causes random, sporadic covalent degradation events leading to local material discontinuity within bulk fibrils. We argue that subsequent fragmentation of the fibrils during sonication (prior to the seeding) occurs preferentially at these mechanically distinct sites which, in turn exposes mostly temperature-modified surfaces that are incompetent for docking interactions with insulin monomers during seeding.