Ultrasonication-induced amyloid fibril formation of beta2-microglobulin.
To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.
Ohhashi
Yumiko
Y
Institute for Protein Research, Osaka University, and CREST, Japan Science and Technology Agency, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Kihara
Miho
M
Naiki
Hironobu
H
Goto
Yuji
Y
D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure*; D052160:Benzothiazoles; D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D018625:Microscopy, Atomic Force; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013010:Sonication; D013844:Thiazoles / Q000737:chemistry*; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000737:chemistry*
D000682:Amyloid; D052160:Benzothiazoles; D011994:Recombinant Proteins; D013844:Thiazoles; D001613:beta 2-Microglobulin; C009462:thioflavin T; D012967:Sodium Dodecyl Sulfate