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首页> 外文期刊>Comments on Inorganic Chemistry: A Journal of Critical Discussion of the Current Literature >THE HELIX-TURN-HELIX AS A SCAFFOLD FOR CHIMERIC NUCLEASE DESIGN
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THE HELIX-TURN-HELIX AS A SCAFFOLD FOR CHIMERIC NUCLEASE DESIGN

机译：HELIX-TURN-HELIX作为嵌合核酸酶设计的支架

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De novo design is a powerful tool to investigate the active site of enzymatic metalloproteins, in a smaller, defined model system. It is also a way to build or combine activity and selectivity in unique ways, not seen biologically. We are utilizing protein design to build artificial endonucleases, and to investigate fundamental questions of metallonuclease structure and function. We have focused on designing peptide constructs comprising geometrically similar turns from unrelated proteins, in particular the Ca-binding EF-hand motif of calmodulin and the helix-turn-helix motif (HTH) of engrailed homeodomain. By substituting the calcium-binding (and thus lan-thanide-binding) loop in place of the "turn" of engrailed HTH, hydrolytically active, DNA-binding constructs were created. The NMR solution structure of one La-binding chimera (P3W), calculated based on NOE volume integrals, demonstrated that the 33-mer peptide retains the parental helix-turn-helix structure when bound to lanthanide ions. The binding affinities of the chimeras for Ln(III) ions are in the low mu M regime, typical for EF-hand sequences, despite the significant changes in flanking sequence. Importantly, the Ln(III) chimeras are catalytically competent, able to hydrolyze phosphate esters including DNA, and were found to bind and cleave DNA with sequence preference. Thus, these designed HTH/EF-hand chimeras represent the first examples of small peptidic artificial nuclease with sequence discrimination, and show that the HTH is a robust scaffold on which to build novel metallopeptide constructs. This review describes the design and characterization of Ln-binding HTH/EF-hand chimeras.
机译：从头设计是一个功能强大的工具，可以在一个较小的定义模型系统中研究酶促金属蛋白的活性位点。这也是一种以生物学上看不到的独特方式建立或结合活性和选择性的方法。我们正在利用蛋白质设计来构建人工核酸内切酶，并研究金属核酸酶的结构和功能的基本问题。我们专注于设计肽构建体，该肽构建体包含来自无关蛋白质的几何相似转折，特别是钙调蛋白的Ca结合EF手基序和出轨同源域的螺旋转折螺旋基序（HTH）。通过取代钙结合（并因此与镧系元素结合）环来代替已结合的HTH的“转变”，就产生了具有水解活性的DNA结合构建体。基于NOE体积积分计算的一种La结合嵌合体（P3W）的NMR溶液结构表明，当33-mer肽与镧系元素离子结合时，该33-mer肽保留了亲本螺旋-转-螺旋结构。尽管侧翼序列发生了显着变化，但嵌合体对Ln（III）离子的结合亲和力处于低μM谱系，这是EF手序列的典型特征。重要的是，Ln（III）嵌合体具有催化能力，能够水解包括DNA的磷酸酯，并发现其具有序列优先结合和切割DNA的能力。因此，这些设计的HTH / EF-手嵌合体代表具有序列区分的小肽人工核酸酶的第一个实例，并且表明HTH是在其上构建新的金属肽构建体的坚固支架。这篇综述描述了Ln结合HTH / EF手嵌合体的设计和表征。

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