We investigated whether any local moieties within a protein molecule could completely refold from the denatured state to regain the native conformation. Bovine β-lactoglobulin (β-LG) was denatured in the presence of guanidine hydrochloride (GdnHCl) as the denaturant. Renaturation of the denatured β- LG was attempted by dialyzing to remove GdnHCl. The renatured molecules regained the same retinol binding activity as that of native β-LG, and physicochemical studies also indicated that refolding of the denatured β-LG had been almost completely successful. Local structural differences between the native and renatured β-LG molecules were evaluated by using our panel of four anti-β-LG monoclonal antibodies (anti-β-LG mAbs). The structures of the epitope regions in native β-LG recognized by two of these mAbs were the same as those in renatured β-LG. However, it is notable that the binding properties of the other two mAbs to native β-LG indicated a wide structural difference in the epitope regions between the native and renatured β-LG. These regions unable to completely refold were the same as those that unfolded preferentially to the α-helix region, shown in the previous report (Kaminogawa, S., Shimizu, M., Ametani, A., Hattori, M., Ando, O., Hachimura, S., Nakamura, Y., Totsuka, M., and Yamauchi, K. (1989) Biochim. Biophys. Acta 998, 50-56). Complete refolding was never attained by several renaturation conditions such as quicker or slower removal of the denaturant, nor by additional oxidation treatment after reducing the disulfide bonds. These results suggest that some specific moiety(ies) in a protein molecule cannot return to the native conformation from a denatured state, even if the other moieties refold completely, and that such a conformational difference between renatured and native forms has no affect on the biological function of ligand binding.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1993|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology