Amyloidogenic potential of alpha-chymotrypsin in different conformational states

Amyloid fibril formation is widely believed to be a generic property of polypeptide chains. In the present study, alpha-chymotrypsin, a well-known serine protease has been driven toward these structures by the use of two different conditions involving (I) high temperature, pH 2.5, and (II) low concentration of trifluoroethanol (TFE), pH 2.5. A variety of experimental methods, including fluorescence emission, dynamic quenching, steady-state fluorescence anisotropy, far-UV circular dichroism, nuclear magnetic resonance spectroscopy, and dynamic light scattering were employed to characterize the conformational states of alpha-chymotrypsin that precede formation of amyloid fibrils. The structure formed under Condition I was an unfolded monomer, whereas an alpha-helical rich oligomer was induced in Condition II. Both the amyloid aggregation-prone species manifested a higher solvent exposure of hydrophobic and aromatic residues compared with the native state. Upon incubation of the protein in these conditions for 48 h, amyloid-like fibrils were formed with diameters of about 10-12 nm. In contrast, at neutral pH and low concentration of TFE, a significant degree of amorphous aggregation was observed, suggesting that charge neutralization of acidic residues in the amyloid core region has a positive influence on amyloid fibril formation. In summary, results presented in this communication suggest that amyloid fibrils of alpha-chymotrypsin may be obtained from a variety of structurally distinct conformational ensembles highlighting the critical importance of protein evolution mechanisms related to prevention of protein misfolding.