Hamada D, Goto Y
Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Japan.
J Mol Biol. 1997 Jun 20;269(4):479-87. doi: 10.1006/jmbi.1997.1055.
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.
一般认为蛋白质折叠中间体包含部分形成的类似天然的二级结构。相比之下，我们最近报道牛β-乳球蛋白的动力学折叠中间体包含非天然的α-螺旋结构。为了理解稳定非天然中间体的机制，我们通过圆二色性（CD）对在pH 2和4℃下盐酸胍（Gdn-HCl）诱导的β-乳球蛋白的平衡去折叠转变进行了表征。近紫外CD测量的去折叠转变先于远紫外CD测量的转变，表明中间体状态的积累。通过对存在不同浓度Gdn-HCl时的CD光谱进行全局拟合分析获得的中间体的远紫外CD光谱，类似于在重折叠动力学中观察到的爆发相中间体，并且包含非天然的α-螺旋结构。添加10%（v/v）的2,2,2-三氟乙醇（TFE）增加了平衡中间体的螺旋含量，尽管在没有Gdn-HCl的情况下蛋白质仍呈现天然结构。构建了构象状态的相图，即α-螺旋中间体、去折叠态和天然态相对于TFE和Gdn-HCl浓度的相图。这表明，由于β-乳球蛋白氨基酸序列的高螺旋偏好性，螺旋区域突出到天然态和去折叠态之间的边界，导致在天然β-折叠结构平衡去折叠时螺旋中间体的非单调积累。这是首次观察到在主要为β-折叠的蛋白质平衡去折叠过程中积累非天然的α-螺旋中间体。
