An engineered azurin variant containing a selenocysteine copper ligand
An engineered azurin variant containing a selenocysteine copper ligand
复制标题
DOI：
10.1021/ja0169163
复制
发表时间：
2002-03-13
影响因子：
15
通讯作者：
Lu, Y
中科院分区：
文献类型：
--
作者：
Berry, SM;Gieselman, MD;Lu, Y
通过重新设计或重新设计现有蛋白质来调节蛋白质的特性一直是蛋白质化学的长期目标。在过去的二十年里，定点诱变已经成为探测某些残基作用和微调蛋白质活性的有力工具。这种方法的一个局限性是，通过遗传密码中的20个氨基酸，只能获得有限数量的官能团。最近的表达蛋白连接（EPL）技术提供了一种替代途径，允许将非天然残基有效地结合到蛋白质中。我们在这里报道了一种蓝铜蛋白变体的制备和光谱表征，其中蓝铜中心的半胱氨酸配体被硒代半胱氨酸EPL取代（Sec）。这是第一次将硒代半胱氨酸人工掺入金属蛋白的活性位点。该变体显示出明显增加的a ||（从56到104 G）和红移的CT波段（从625到677 nm），同时保持了一般的1型铜特征，包括还原电位的相似性。这项研究表明，使用EPL的同位结构取代可以微调金属结合位点的结构和功能性质，而不会损失其大部分特征。进一步的光谱和x射线晶体学研究将为金属蛋白的结构和功能的精细控制提供新的见解。
Modulating the properties of proteins through de novo design or redesign of existing proteins has been a longstanding goal in protein chemistry. Over the past two decades, site-directed mutagenesis has been a powerful tool to probe the role of certain residues and to fine-tune the activity of proteins. A limitation of this approach has been the accessibility of only a restricted number of functional groups through the 20 amino acids in the genetic code. The more recent technique of expressed protein ligation (EPL) provides an alternative route that allows efficient incorporation of nonnatural residues into proteins. We report here the preparation and spectroscopic characterization of an azurin variant in which a cysteine ligand to the blue copper center has been replaced by EPL with selenocysteine (Sec). This reports marks the first time that selenocysteine is artificially incorporated into the active site of a metalloprotein. The variant displays a significantly increased A|| (from 56 to 104 G) and red-shifted CT band (from 625 to 677 nm), while maintaining the general type 1 copper characteristics, including similarity in reduction potentials. This study illustrates that iso-structural substitution using EPL can fine-tune the structural and functional properties of a metal-binding site without loss of most of its characteristics. Further spectroscopic and X-ray crystallographic studies of this and other EPL variants will provide new insights into the fine-control of the structure and function of metalloproteins.