Sanz J M, Johnson C M, Fersht A R
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, Medical Research Council Centre, U.K.
Biochemistry. 1994 Sep 20;33(37):11189-99. doi: 10.1021/bi00203a015.
The acid-induced denaturation of barnase and its mutants has been analyzed to search for partly-folded intermediates. Differential scanning calorimetry of barnase deviates from two-state behavior below pH 4.0 at low ionic strength, with the maximum discrepancy at pH 2.7. Addition of 200 mM KCl apparently restores the two-state transitions. Thermograms of barnase mutants at pH 2.7 and low ionic strength fall into three classes: alpha, symmetric transitions which fit well to a two-state equilibrium; b, asymmetric transitions indicating deviation from two-state behavior; and c, transitions with an obvious second component. The most distorted thermograms are observed for mutants that had previously been engineered to accumulate at equilibrium the major kinetic folding intermediate state of barnase at neutral pH. Further analysis of these mutants show the existence of complex equilibria on thermal denaturation. Addition of KCl leads to the slow formation of soluble aggregated forms (A-state) which share some of the properties of the "molten globule" state, i.e., significant secondary structure, lack of fixed tertiary structure, and solvent-accessible hydrophobic patches. The far-UV CD spectrum of the A-state can be explained in terms of native-like secondary structure contributions. Kinetic and chemical cross-linking experiments show that dimerization of partly-folded molecules occurs in the transition region, and such dimerization is probably the rate-limiting step in the formation of the A-state in the presence of KCl. As the A-state has been observed clearly so far for only the mutants in which the folding intermediate has been designed to accumulate, we suggest that the A-state would be related to the main folding intermediate state of barnase. The intermediate would be highly stabilized at low pH, and it is prone to self-associate in these conditions.
为了寻找部分折叠中间体，对芽孢杆菌RNA酶及其突变体的酸诱导变性进行了分析。在低离子强度下，pH值低于4.0时，芽孢杆菌RNA酶的差示扫描量热法偏离了两态行为，在pH 2.7时差异最大。添加200 mM KCl显然可恢复两态转变。在pH 2.7和低离子强度下，芽孢杆菌RNA酶突变体的热谱图分为三类：a，对称转变，很好地符合两态平衡；b，不对称转变，表明偏离两态行为；c，具有明显第二成分的转变。对于先前经设计在中性pH下平衡积累芽孢杆菌RNA酶主要动力学折叠中间态的突变体，观察到最扭曲的热谱图。对这些突变体的进一步分析表明，热变性时存在复杂的平衡。添加KCl会导致缓慢形成可溶性聚集形式（A态），其具有一些“熔球”态的特性，即显著的二级结构、缺乏固定的三级结构以及溶剂可及的疏水斑块。A态的远紫外圆二色光谱可用类似天然的二级结构贡献来解释。动力学和化学交联实验表明，部分折叠分子在转变区域发生二聚化，并且这种二聚化可能是在KCl存在下形成A态的限速步骤。由于到目前为止仅在设计用于积累折叠中间体的突变体中清楚地观察到了A态，我们认为A态与芽孢杆菌RNA酶的主要折叠中间态有关。该中间态在低pH下会高度稳定，并且在这些条件下易于自缔合。
