Hattori M, Ametani A, Katakura Y, Shimizu M, Kaminogawa S
Department of Agricultural Chemistry, University of Tokyo, Japan.
J Biol Chem. 1993 Oct 25;268(30):22414-9.
We investigated whether any local moieties within a protein molecule could completely refold from the denatured state to regain the native conformation. Bovine beta-lactoglobulin (beta-LG) was denatured in the presence of guanidine hydrochloride (GdnHCl) as the denaturant. Renaturation of the denatured beta-LG was attempted by dialyzing to remove GdnHCl. The renatured molecules regained the same retinol binding activity as that of native beta-LG, and physicochemical studies also indicated that refolding of the denatured beta-LG had been almost completely successful. Local structural differences between the native and renatured beta-LG molecules were evaluated by using our panel of four anti-beta-LG monoclonal antibodies (anti-beta-LG mAbs). The structures of the epitope regions in native beta-LG recognized by two of these mAbs were the same as those in renatured beta-LG. However, it is notable that the binding properties of the other two mAbs to native beta-LG indicated a wide structural difference in the epitope regions between the native and renatured beta-LG. These regions unable to completely refold were the same as those that unfolded preferentially to the alpha-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.
我们研究了蛋白质分子内的任何局部部分是否能够从变性状态完全重新折叠以恢复天然构象。牛β-乳球蛋白（β-LG）在作为变性剂的盐酸胍（GdnHCl）存在下变性。通过透析去除GdnHCl来尝试使变性的β-LG复性。复性后的分子恢复了与天然β-LG相同的视黄醇结合活性，并且物理化学研究也表明变性的β-LG的重新折叠几乎完全成功。使用我们的四种抗β-LG单克隆抗体（抗β-LG mAb）对天然和复性的β-LG分子之间的局部结构差异进行了评估。其中两种mAb识别的天然β-LG中表位区域的结构与复性β-LG中的相同。然而，值得注意的是，另外两种mAb与天然β-LG的结合特性表明天然和复性β-LG之间表位区域存在广泛的结构差异。这些不能完全重新折叠的区域与先前报告（Kaminogawa，S.，Shimizu，M.，Ametani，A.，Hattori，M.，Ando，O.，Hachimura，S.，Nakamura，Y.，Totsuka，M.和Yamauchi，K.（1989）Biochim. Biophys. Acta 998，50 - 56）中优先于α-螺旋区域展开的区域相同。通过几种复性条件，如更快或更慢地去除变性剂，以及在还原二硫键后进行额外的氧化处理，都从未实现完全重新折叠。这些结果表明，蛋白质分子中的某些特定部分即使其他部分完全重新折叠也不能从变性状态恢复到天然构象，并且复性和天然形式之间的这种构象差异对配体结合的生物学功能没有影响。
