Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids
Probing the role of axial methionine in the blue copper center of azurin with unnatural amino acids
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DOI：
10.1021/ja029699u
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发表时间：
2003-07-23
影响因子：
15
通讯作者：
Lu, Y
中科院分区：
文献类型：
--
作者：
Berry, SM;Ralle, M;Lu, Y
利用表达蛋白连接技术将铜绿假单胞菌蓝铜蛋白天青蛋白的轴向蛋氨酸替换为非天然氨基酸。高度保守的蛋氨酸121残基被替换为同结构氨基酸正亮氨酸（NIe）和硒代蛋氨酸（SeM）。的紫外-可见吸收，X-和Q-波段EPR，和Cu EXAFS光谱的变体从WT略有扰动。所有的变体都有一个主要的S（Cys）到Cu（II）的电荷转移带约625 nm和窄EPR超精细分裂。还报道了M121 SeM变体的Se EXAFS。与小的光谱变化相反，M121 SeM、M121 Leu和M121 NIe的还原电位分别比WT天青蛋白高25、135和140 mV。非天然氨基酸的使用允许对影响蓝铜中心的还原电位的不同因素进行解卷积。对WT天青蛋白及其变体的仔细分析表明，大的还原电位变化与轴向配体侧链的疏水性线性相关。因此，疏水性是调节轴向配体对蓝铜中心还原电位的主导因素。
Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (NIe) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121NIe are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.