Crystal structure and preliminary functional analysis of the cytochrome c peroxidase His175Gln proximal ligand mutant
Crystal structure and preliminary functional analysis of the cytochrome c peroxidase His175Gln proximal ligand mutant
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细胞色素c过氧化物酶His175Gln近端配体突变体的晶体结构和初步功能分析
DOI：
10.1021/ja00020a044
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发表时间：
1991
影响因子：
15
通讯作者：
T. Poulos
中科院分区：
文献类型：
--
作者：
M. Sundaramoorthy;K. Choudhury;S. Edwards;T. Poulos
晶体学计算，Diamond, R. 等人编辑；印度科学研究所：印度班加罗尔，1980 年； pp 13.1-13.23）用于精修。‘R“£| F0-Fcl/ERo·‘最终模型的均方根偏差代表键距与预期值的均方根偏差。有助于将血红素固定到位。为了直接测试近端配体的作用，我们开发了 CCP 的重组大肠杆菌表达系统。4 该系统已用于转换近端配体，使用定点诱变将重组野生型和 Hisl75Gln 突变体纯化并结晶。 5 突变体的可见吸收光谱类似于高自旋光谱，其索雷特最大值与 280 nm 的比率为 1.25，这与野生型酶的比率为 1.3 相当，表明如果血红素是六配位的，则它在 pH 5.5 时仍保持高自旋。 pH 5.5 下的吸收光谱与 (4) Darwish, K.；Li, H.；Poulos, T. L. Protein Eng.，出版中的吸收光谱非常相似。(5) 重组 CCP 的纯化按照 Fishel 等人的方法进行（Fishel, LA；Villafranca, J. E.；Mauro, J. M.；Kraut, J. Biochemistry 1987, 26, 351-360），并稍作修改。根据 Edwards 和 Poulos (Edwards, S. E.; Poulos, TLJ Biol. Chem. 1990, 265, 2588-2595) 的方法，用 2-甲基-2, 4-戊二醇 (MPD) 制备晶体，并进行以下修改，加入约 16 pL 10 mg/mL CCP，溶于 0.05 M 磷酸钾，pH 6.0，并且25% MPD 被输送到含有小晶种的 1 毫米宽的 X 射线毛细管中，晶种已被清洗，从 32.5% 开始，到 27.5% 结束，增量约为 2.5%。毛细管被密封在一个夹层盒中，并在冷室 (4°C) 中对 35% MPD 进行蒸气扩散。液体塞重新引入晶体上方，并且用矿物油密封的毛细管被证明是对温度敏感的，因此所有数据集都是在2至5°C之间获得的。
Computing in Crystallography·, Diamond, R., et al., Eds.; Indian Institute of Science: Bangalore, India, 1980; pp 13.1-13.23) was used for the re-finement.‘R “£| F0-Fcl/ERo·‘The rms deviation of the final model represents the root-mean-square deviation of bond distances from expected values. helping to hold the heme in place. In order to directly test the role of the proximal ligand, we have developed a recombinant Escherichia coli expression system for CCP. 4 This system has been used toconvert the proximal ligand, Hisl75, to Gin using site-directed mutagenesis. Both recombinant wild type and the Hisl75Gln mutant were purified and crystallized. 5 The visible absorption spectrum of the mutant resembles a high-spin spectrum with a Soret maximum to 280 nm ratio of 1.25, which is com-parable to the wild type enzyme ratioof 1.3, indicating that if the heme is hexacoordinate, it remains high-spin atpH 5.5. While the absorption spectrum at pH 5.5 is very similar to that of (4) Darwish, K.; Li, H.; Poulos, T. L. Protein Eng., in press.(5) Purification of recombinant CCP was according to Fishel et al.(Fishel, LA; Villafranca, J. E.; Mauro, J. M.; Kraut, J. Biochemistry 1987, 26, 351-360) with slight modifications. Crystals were prepared from 2-methyl-2, 4-pentanediol (MPD) according to Edwards and Poulos (Edwards, S. E.; Poulos, TLJ Biol. Chem. 1990, 265, 2588-2595) with the following modifications. Approximately 16 pL of 10 mg/mL CCP in 0.05 M potassium phosphate, pH 6.0, and 25% MPD was delivered into a 1-mm-wide X-ray capillary containing a small seed crystal. The seed crystal hadbeen washed previously with decreasing concentrations of MPD beginning with 32.5% and ending with 27.5% in approximately 2.5% increments. The capillary was sealed in a sandwich box and vapor diffused against 35% MPD in the cold room (4 C). Once the crystal had grown to sufficient size, the remainder of the surrounding solution was carefully removed, a mother liquor plug reintroduced above the crystal, and the capillary sealed with mineral oil. Mutant crystalsproved to be temperature sensitive, so all data sets were obtained between 2 and 5 C.