Trifluoroethanol-induced Stabilization of the α-Helical Structure of β-Lactoglobulin: Implication for Non-hierarchical Protein Folding

Abstract

Trifluoroethanol (TFE) is known to stabilize the α-helical structure in proteins and their fragments. However, the relationship between the TFE-induced structures and the native structure is not clear. Here we show that β-lactoglobulin, which consists predominantly of β-sheets, exhibited a markedly high propensity to form an α–-helical structure in the presence of TFE, as measured by far-UV circular dichroism. A cooperative transformation from the β-sheet structure to an α-helical structure occurred at a TFE concentration between 10% and 20%. These results were in contrast to a gradual β-sheet to α-helix transition of the constant fragment of the immunoglobulin light chain, which is also a β-sheet protein. To understand the significance of the high helical propensity of β-lactoglobulin we measured the LTFE-induced conformational transition of more than 20 proteins of various secondary structural types. Whereas the α-helical proteins showed a propensity to form an extensive helical structure in TFE, the helical propensity of proteins with a low helical content in the native state varied. The helical content in TFE was correlated more with the helical content predicted by a secondary structure prediction than with the helical content of the native structure, suggesting that the stability of the helical structure in TFE is determined by local interactions between nearby amino acid residues. Our results suggest that an α-helical intermediate can accumulate during the refolding process of β-lactoglobulin and that a hierarchical model of protein folding is not necessarily true for some β-sheet proteins including β-lactoglobulin.