Creation of a Binuclear Purple Copper Site within a de Novo Coiled-Coil Protein
Creation of a Binuclear Purple Copper Site within a de Novo Coiled-Coil Protein
复制标题
在 de Novo 卷曲螺旋蛋白内创建双核紫铜位点
DOI：
10.1021/bi3007884
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
T. Tanaka
中科院分区：
文献类型：
--
作者：
D. Shiga;Y. Funahashi;H. Masuda;A. Kikuchi;M. Noda;S. Uchiyama;K. Fukui;K. Kanaori;K. Tajima;Y. Takano;H. Nakamura;M. Kamei;T. Tanaka
尽管已经通过从头设计构建了各种金属结合蛋白，但双核金属结合位点的创建仍然特别具有挑战性。 COX 亚基 II 中的紫铜位点（称为 CuA位点）具有由两个 Cys 残基桥接的两个铜离子。我们在重新设计的四螺旋卷曲螺旋蛋白中构建了由两个半胱氨酸和两个组氨酸残基组成的 CuA 位点。该蛋白质结合了两个铜离子并呈现紫色，在紫外可见光谱中在 488 和 530 nm 处有相对较强的吸收带。 EPR 光谱显示 g∥ 区域中未解决的超精细分裂，这与 A∼480/A∼530> 1 的天然或工程 CuAsite 类似。蛋白质的扩展 X 射线吸收结构分析揭示了 Cu2S2 核心结构的存在，每个核心有两个典型的 N(His)-Cu 键，位于 1.90 Å，两个 S (Cys)-Cu 键位于 2.21 Å，并且Cu-Cu键位于2.51 Å处，这也是紫铜位点的特征结构。
Although various kinds of metal binding proteins have been constructed byde novodesign, the creation of a binuclear metal binding site remains especially challenging. The purple copper site in subunit II of COX, referred to as the CuAsite, has two copper ions bridged by two Cys residues. We constructed the CuAsite consisting of two Cys and two His residues in ade novodesigned four-helical coiled-coil protein. The protein bound two copper ions and exhibited a purple color, with relatively intense absorption bands at 488 and 530 nm in the UV–vis spectrum. The EPR spectrum displayed unresolved hyperfine splittings in theg∥region, which was similar to the native or engineered CuAsite with anA∼480/A∼530> 1. The extended X-ray absorption structure analyses of the protein revealed the presence of the Cu2S2core structure, with two typical N(His)–Cu bonds per core at 1.90 Å, two S (Cys)–Cu bonds at 2.21 Å, and the Cu–Cu bond at 2.51 Å, which are also characteristic structures of a purple copper site.