Amyloid fibril formation by a partially structured intermediate state of alpha-chymotrypsin.
Here we investigated the effects of 2,2,2-trifluoroethanol (TFE) on the structure of alpha-chymotrypsin. The protein aggregates maximally in 35% (v/v) TFE. Congo red and thioflavin-T binding experiments suggest that the aggregates induced by TFE have amyloid-like properties, and transmission electron microscopy data show that these aggregates have a fibrilar morphology. Fluorescence, circular dichroism, anilino-8-napthalene sulfonate binding, and Fourier-transformed infrared spectroscopy data suggest that formation of a partially structured intermediate state precedes the onset of the aggregation process. The native beta-barrel structure of alpha-chymotrypsin appears to be disrupted in the partially structured intermediate state in favour of a non-native extended beta-sheet conformation with exposed hydrophobic surfaces. The protein becomes "sticky" under these conditions and aggregates into amyloid-like structures. The data support the hypothesis that amyloid formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrilar aggregates.
Pallarès
Irantzu
I
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
Vendrell
Josep
J
Avilés
Francesc X
FX
Ventura
Salvador
S
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D052160:Benzothiazoles; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002942:Circular Dichroism; D003224:Congo Red / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000378:metabolism; D014270:Trifluoroethanol / Q000737:chemistry*
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red; D014270:Trifluoroethanol; D002918:Chymotrypsin; C117608:alpha-chymotrypsin