Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 342, Issue 1, Pages 321-331Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2004.06.089
Keywords
amyloid formation; alpha-chymotrypsin; protein misfolding; TFE-induced protein denaturation; aggregation intermediates; serine proteases
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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. (C) 2004 Elsevier Ltd. All rights reserved.
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