THE EQUILIBRIUM INTERMEDIATE OF BETA-LACTOGLOBULIN WITH NONNATIVE ALPHA-HELICAL STRUCTURE

It is generally considered that intermediates of protein folding conta in partially formed native-like secondary structures. In contrast, we recently reported that the kinetic folding intermediate of bovine beta -lactoglobulin contains non-native alpha-helical structures. To unders tand the mechanism that stabilizes the non-native intermediate, we cha racterized by circular dichroism (CD) the equilibrium unfolding transi tion of beta-lactoglobulin induced by guanidine hydrochloride (Gdn-HCl ) at pH 2 and 4 degrees C. The unfolding transition measured by near-U V CD preceded the transition measured by far-UV CD, indicating the acc umulation of the intermediate state. The far-UV CD spectrum of the int ermediate, obtained by global fitting analysis of the CD spectra in th e presence of various concentrations of Gdn-HCl, was similar to the bu rst-phase intermediate observed in the refolding kinetics, and contain ed non-native alpha-helical structures. Addition of 10% (v/v) 2,2,2-tr ifluoroethanol (TFE) increased the helical content of the equilibrium intermediate, although the protein still assumed the native structure in the absence of Gdn-HCl. A phase diagram of the conformational state s, i.e. the alpha-helical intermediate, unfolded and native states, ag ainst the concentration of TFE and Gdn-HCl was constructed. This indic ated that, because of the high helical preference of the amino add seq uence of beta-lactoglobulin, the helical region protrudes into the bou ndary between the native and unfolded states, resulting in non-monoton ic accumulation of the helical intermediate upon equilibrium unfolding of the native beta-sheet structure. This is the first observation to indicate that a non-native alpha-helical intermediate accumulates duri ng equilibrium unfolding of a predominantly beta-sheet protein. (C) 19 97 Academic Press Limited.