Regan L, Clarke N D
Laboratory of Molecular Biology, Medical Research Council, Cambridge, U.K.
Biochemistry. 1990 Dec 11;29(49):10878-83. doi: 10.1021/bi00501a003.
The ultimate goal of protein engineering is to create novel proteins which will adopt predetermined structures, bind specified ligands, and catalyze new reactions. Here we describe the successful introduction of metal-binding activity into a model four helix bundle protein. The designed binding site is tetrahedral and is formed by two Cys and two His ligands on adjacent helices. We have introduced this site into the protein and characterized the binding activity. Using 65Zn(II), we have shown that the protein binds Zn(II), that the sulfhydryls are essential for binding, and that binding occurs to the protein monomer. The designed protein binds metals with high affinity: we estimate the dissociation constants as 2.5 X 10(-8) M for Zn(II) and 1.6 X 10(-5) M for Co(II). The characteristic absorption spectrum of the Co(II)-substituted protein fully supports the model of a tetrahedral binding site comprised of two Cys and two His ligands. Circular dichroism studies indicate that no significant changes in secondary structure occur between the metal-bound and metal-free forms of the protein. However, the metal-bound form is substantially stabilized toward denaturation by GuHCl compared to the metal-free form.
蛋白质工程的最终目标是创造出能呈现预定结构、结合特定配体并催化新反应的新型蛋白质。在此，我们描述了如何成功地将金属结合活性引入一种典型的四螺旋束蛋白中。所设计的结合位点呈四面体结构，由相邻螺旋上的两个半胱氨酸（Cys）和两个组氨酸（His）配体构成。我们已将该位点引入蛋白并对其结合活性进行了表征。利用65Zn（II），我们证明了该蛋白能结合Zn（II），巯基对于结合至关重要，且结合发生在蛋白单体上。所设计的蛋白以高亲和力结合金属：我们估计Zn（II）的解离常数为2.5×10^(-8) M，Co（II）的解离常数为1.6×10^(-5) M。Co（II）取代蛋白的特征吸收光谱充分支持了由两个半胱氨酸和两个组氨酸配体组成的四面体结合位点模型。圆二色性研究表明，蛋白结合金属和未结合金属的形式之间二级结构没有显著变化。然而，与未结合金属的形式相比，结合金属的形式对盐酸胍（GuHCl）诱导的变性具有显著更高的稳定性。
