The equilibrium intermediate of beta-lactoglobulin with non-native alpha-helical structure.
It is generally considered that intermediates of protein folding contain 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 understand the mechanism that stabilizes the non-native intermediate, we characterized by circular dichroism (CD) the equilibrium unfolding transition of beta-lactoglobulin induced by guanidine hydrochloride (Gdn-HCl) at pH 2 and 4 degrees C. The unfolding transition measured by near-UV CD preceded the transition measured by far-UV CD, indicating the accumulation of the intermediate state. The far-UV CD spectrum of the intermediate, obtained by global fitting analysis of the CD spectra in the presence of various concentrations of Gdn-HCl, was similar to the burst-phase intermediate observed in the refolding kinetics, and contained non-native alpha-helical structures. Addition of 10% (v/v) 2,2,2-trifluoroethanol (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 states, i.e. the alpha-helical intermediate, unfolded and native states, against the concentration of TFE and Gdn-HCl was constructed. This indicated that, because of the high helical preference of the amino acid sequence of beta-lactoglobulin, the helical region protrudes into the boundary between the native and unfolded states, resulting in non-monotonic 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 during equilibrium unfolding of a predominantly beta-sheet protein.
Hamada
D
D
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Japan.
Goto
Y
Y
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007782:Lactoglobulins / Q000737:chemistry* / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D006146:Guanidines; D007782:Lactoglobulins; D019791:Guanidine