High-throughput analysis of the ultrasonication-forced amyloid fibrillation reveals the mechanism underlying the large fluctuation in the lag time [Protein Structure and Folding]

August 12th, 2014 by Umemoto, A., Yagi, H., So, M., Goto, Y.

Amyloid fibrils form in supersaturated solutions of precursor proteins by a nucleation and growth mechanism characterized by a lag time. Although the lag time provides a clue to understanding the complexity of nucleation events, its long period and low reproducibility have been obstacles for exact analysis. Ultrasonication is known to effectively break supersaturation and force fibrillation. By constructing a HANdai Amyloid Burst Inducer (HANABI), which combines the use of a water-bath-type ultrasonicator and microplate reader, we examined the ultrasonication-forced fibrillation of several proteins with a focus on the fluctuation in the lag time. Amyloid fibrillation of hen egg white lysozyme was examined at pH 2.0 in the presence of 1.0-5.0 M guanidium hydrochloride (Gdn-HCl) in which the dominant species varied from the native to denatured conformations. Although fibrillation occurred at various concentrations of Gdn-HCl, the lag time varied largely with a minimum being observed at approximately 3.0 M, the concentration at which Gdn-HCl-dependent denaturation ended. The coefficient of variation of the lag time did not depend significantly on the Gdn-HCl concentration and was two-fold larger than that of the ultrasonication-dependent oxidation of iodide, a simple model reaction. These results suggest that the large fluctuation observed in the lag time for amyloid fibrillation originated from a process associated with a common amyloidogenic intermediate, which may have been a relatively compact denatured conformation. We also suggest that the HANABI system will be useful for studying the mechanism of crystallization of proteins because proteins form crystals by the same mechanism as amyloid fibrils under supersaturation.
  • Posted in Journal of Biological Chemistry, Publications
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