Fig. 4-6 Phase diagram and schematic diagram of the pathway of the amyloid fibril formation of hen egg white lysozyme A phase diagram of the structural states of hen egg white lysozyme is shown as a function of the protein concentrations and ethanol concentrations. This diagram was obtained from the analysis of X-ray and neutron small-angle scattering experiments. It was inferred from the schematic diagram that the amyloid fibril formation pathway proceeds through the steps of dimer formation, protofilament formation, and amyloid fibril formation.

Fig. 4-7 Temporal changes of the structural parameters in the process of amyloid fibril formation of hen egg white lysozyme obtained from time-resolved neutron scattering experiments The radius of gyration of the cross section is related to the shape of the cross section of the filaments, IX(0)/c, which in turn is proportional to the mass per unit length of the filament, and the integrated intensity is related to the association states of the filaments. From these temporal changes of structural parameters obtained from the analysis of neutron scattering curves, it was found that amyloid fibril formation by which the cross section of the fibrils becomes large, and the association of the amyloid fibrils that does not change the cross-section of the fibrils, occur sequentially.

"Amyloid" diseases including various neuro-degerative diseases such as Alzheimer's disease and bovine spongiform encephalopathy (BSE, the so-called mad cow disease) share characteristics that misfolded proteins form filamentous structures called the amyloid fibrils and these fibrils are accumulated as deposits in tissues and organs. Recently, it has become known that not only the proteins related to the amyloid diseases but also a wide variety of proteins that are not related to any diseases form amyloid fibrils. Understanding the mechanism of the amyloid fibril formation is not only important for the development of therapeutic strategies against the amyloid diseases, but also is of considerable help to gain an insight into the generic properties of proteins. We employed hen egg white lysozyme (HEWL) as a model protein to study the mechanism of the amyloid fibrils and to investigate the process of the amyloid fibril formation of HEWL in ethanol solution with X-ray and neutron small-angle scattering. Small-angle scattering is an important method to study gross shapes and association states of particles in solution. We performed X-ray and neutron small-angle scattering experiments of HEWL in various HEWL concentrations and in various ethanol concentrations. From the results of these experiments, a phase diagram of the amyloid fibril formation was obtained, and it was shown that the amyloid fibril formation of HEWL occurs through the formation of the dimers, the formation of the protofilaments, and the formation of the amyloid fibrils by lateral association of the protofilaments (Fig. 4-6). We also investigated temporal changes of the structural states of HEWL in the process of the amyloid fibril formation with time-resolved neutron scattering. From the temporal changes of the structural parameters obtained, it was shown that association of the amyloid fibrils occurs following the formation of the amyloid fibrils by the association of the protofilaments (Fig. 4-7). It is expected that understanding the mechanism of the amyloid fibril formation with neutron and X-ray small-angle scattering will ultimately contribute to understanding the mechanism of the amyloid diseases and their therapeutic strategy.

Reference S. Fujiwara et al., Effects of Salt Concentration on Association of the Amyloid Protofilaments of Hen Egg White Lysozyme Studied by Time-resolved Neutron Scattering, J. Mol. Biol., 331, 21 (2003).

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