Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution.
Mutant human lysozymes (Ile56Thr & Asp67His) have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a beta-structure from the native alpha-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg/mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of approximately 70 A. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 A, a reflection that arises from the interstrand spacing in beta-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.
Goda
S
S
Institute for Protein Research, Osaka University, Suita, Japan.
Takano
K
K
Yamagata
Y
Y
Nagata
R
R
Akutsu
H
H
Maki
S
S
Namba
K
K
Yutani
K
K
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D000431:Ethanol; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008854:Microscopy, Electron; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D012996:Solutions; D014961:X-Ray Diffraction
D000682:Amyloid; D012996:Solutions; D000431:Ethanol; D009113:Muramidase