De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1
De novo design of a D2-symmetrical protein that reproduces the diheme four-helix bundle in cytochrome bc1
复制标题
DOI：
10.1021/ja039935g
复制
发表时间：
2004-07-07
影响因子：
15
通讯作者：
DeGrado, WF
中科院分区：
文献类型：
--
作者：
Ghirlanda, G;Osyczka, A;DeGrado, WF
基于细胞色素 bc(1) 中跨膜二血红四螺旋束主链坐标的数学参数化，构建了理想化的水溶性 D-2 对称二血红蛋白。每个血红素由对角螺旋的两个 His 残基协调。在该模型中，His 配体的咪唑环保持在有点不寻常的垂直方向，如细胞色素 bc(1) 中所见，该方向通过与相邻螺旋上的 Thr 侧链的第二壳层氢键维持。所得肽在脱辅基状态下展开，但在与血红素结合后协同组装成折叠良好的四聚体。每个四聚体在低微摩尔浓度下以高亲和力结合两个血红素。平衡还原中点电位相对于 SHE 在还原和氧化方向上分别在 -76 mV 和 -124 mV 之间变化。三价铁络合物的 EPR 谱表明存在低自旋物质，gm x 值为 3.35，与细胞色素 bc(1) 的血红素 b 获得的值相当（3.79 和 3.44）。这为咪唑配体的垂直取向设计提供了强有力的支持。此外，核磁共振谱表明该蛋白质以独特的构象存在于溶液中，适合结构研究。该蛋白质可以为确定第二壳配体如何影响血红素辅因子的氧化还原电位提供有用的支架。
An idealized, water-soluble D-2-symmetric diheme protein is constructed based on a mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(1). Each heme is coordinated by two His residues from diagonally apposed helices. In the model, the imidazole rings of the His ligands are held in a somewhat unusual perpendicular orientation as found in cytochrome bc(1), which is maintained by a second-shell hydrogen bond to a Thr side chain on a neighboring helix. The resulting peptide is unfolded in the apo state but assembles cooperatively upon binding to heme into a well-folded tetramer. Each tetramer binds two hemes with high affinity at low micromolar concentrations. The equilibrium reduction midpoint potential varies between -76 mV and -124 mV vs SHE in the reducing and oxidizing direction, respectively. The EPR spectrum of the ferric complex indicates the presence of a low-spin species, with a gm x value of 3.35 comparable to those obtained for hemes b of cytochrome bc(1) (3.79 and 3.44). This provides strong support for the designed perpendicular orientation of the imidazole ligands. Moreover, NMR spectra show that the protein exists in solution in a unique conformation and is amenable to structural studies. This protein may provide a useful scaffold for determining how second-shell ligands affect the redox potential of the heme cofactor.