Conformation of beta 2-microglobulin amyloid fibrils analyzed by reduction of the disulfide bond.
Beta2-microglobulin (beta2-m), a major component of dialysis-related amyloid fibrils, has an intrachain disulfide bond buried inside the native structure. We examined the conformation of beta2-m amyloid fibrils by analyzing the reactivity of the disulfide bond to a reducing reagent, dithiothreitol. Although the disulfide bond in the native structure was highly protected from reduction, the disulfide bonds in the amyloid fibrils prepared at pH 2.5 were progressively reduced at pH 8.5 by 50 mm dithiothreitol. Because beta2-m amyloid fibrils prepared under acidic conditions have been known to depolymerize at a neutral pH, we examined the relation between depolymerization and reduction of the disulfide bond. The results indicate that the disulfide bonds in the amyloid fibrils were protected from reduction, and the reduction occurred during depolymerization. On the other hand, the disulfide bonds of immature filaments, the thin and flexible filaments prepared under conditions of high salt at pH 2.5, were reduced at pH 8.5 more readily than those of amyloid fibrils, suggesting that the disulfide bonds are exposed to the solvent. Taken together, the disulfide bond once exposed to the solvent upon acid denaturation may be progressively buried in the interior of the amyloid fibrils during its formation.
Hong
Dong-Pyo
DP
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Gozu
Masayo
M
Hasegawa
Kazuhiro
K
Naiki
Hironobu
H
Goto
Yuji
Y
D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry*
D000682:Amyloid; D052160:Benzothiazoles; D004220:Disulfides; D011994:Recombinant Proteins; D013844:Thiazoles; D001613:beta 2-Microglobulin; C009462:thioflavin T; D003545:Cysteine