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首页> 外文期刊>Biochemistry >Site-Specific Chemical Labeling of Mitochondrial Respiratory Complex I through Ligand-Directed Tosylate Chemistry
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Site-Specific Chemical Labeling of Mitochondrial Respiratory Complex I through Ligand-Directed Tosylate Chemistry

机译：线粒体呼吸道络合物I通过配体定向甲磺酸根化学的特异性化学品标记

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摘要

The site-specific chemical modification of NADH-quinone oxidoreductase (complex I) by various functional probes such as fluorophores and microbeads, without affecting the enzyme activity, may allow singlemolecule analyses of putative dynamic conformational changes in the enzyme. In an attempt to address this challenge, we performed site-specific alkynylation of complex I in bovine heart submitochondrial particles by means of a ligand-directed tosylate (LDT) chemistry strategy with synthetic acetogenin ligand 1, which has an alkynylated tosylate in the tail moiety, as a high-affinity ligand against the enzyme. The terminal alkyne was chosen as the tag to be incorporated into the enzyme because this functional group can serve as a “footing” for subsequent diverse chemical modifications via so-called click chemistry (i.e., azide?alkyne [3+2] cycloaddition in water). To identify the position alkynylated by ligand 1, fluorescent tetramethylrhodamine was covalently attached to the incorporated alkyne by click chemistry after the solubilization of complex I. Detailed proteomic analyses revealed that alkynylation occurred at Asp160 in the 49 kDa subunit, which may be located in the inner part of the putative quinone-binding cavity. The alkynylation was completely suppressed in the presence of an excess of other inhibitors such as bullatacin and quinazoline. While the reaction yield of the alkynylation step via LDT chemistry was estimated to be ～50%, the alkynylation unfortunately resulted in the almost complete inhibition of enzyme activity. Nevertheless, the results of this study demonstrate that complex I can be site-specifically alkynylated through LDT chemistry, providing a clue about the diverse chemical modifications of the enzyme in combination with click chemistry.
机译：通过各种功能探针如荧光团和微珠（荧光团和微珠）的特异性特异性化学改性，例如荧光团和微珠，而不影响酶活性，可以允许酶中推定的动态构象变化的单分子分析。为了解决这一挑战，我们通过具有合成乙酰丙酮（LDT）化学配体1的配体 - 引导的甲磺酸盐（LDT）化学策略在牛心脏提取物颗粒中对复合物I的基本特异性蛋白质进行了特异性蛋白质，其在尾部中具有醇​​烷基化甲苯磺酸盐，作为对酶的高亲和力配体。选择末端炔烃作为待掺入酶中的标签，因为该官能团可用作随后通过所谓的咔哒化学（即叠氮化物β炔烃[3 + 2]环加成在水中的“基于水” ）。为了鉴定配体1的蛋白质蛋白质的位置，在复合物I的溶解后，通过点击化学将荧光四甲基溴胺共价连接到已掺入的炔烃上。详细的蛋白质组学分析显示，在49kDa亚基的Asp160中发生醇烷基化，其可以位于内部推定的醌绑定腔的一部分。在过量的其他抑制剂存在如牛肉蛋白和喹唑啉的情况下，炔基化完全抑制。虽然通过LDT化学的醇烷化步骤的反应产率估计为约50％，但似乎不幸的是导致酶活性的几乎完全抑制。然而，该研究的结果表明，通过LDT化学方法，可以将络合物特异性醇化，提供关于酶的多样化化学修饰的线索与点击化学。

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  • 来源
    《Biochemistry》 |2014年第14期|共11页
  • 作者

    Takahiro Masuya; Masatoshi Murai; Kentaro Ifuku; Hironobu Morisaka; Hideto Miyoshi;

  • 作者单位

    Division of Applied Life Sciences Graduate School of Agriculture and Sakyo-ku Kyoto 606-8502 Japan;

    Division of Applied Life Sciences Graduate School of Agriculture and Sakyo-ku Kyoto 606-8502 Japan;

    Division of Integrated Life Science Graduate School of Biostudies Kyoto University Sakyo-ku Kyoto 606-8502 Japan;

    Division of Applied Life Sciences Graduate School of Agriculture and Sakyo-ku Kyoto 606-8502 Japan;

    Division of Applied Life Sciences Graduate School of Agriculture and Sakyo-ku Kyoto 606-8502 Japan;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Site-Specific; Chemical; Labeling;

    机译：特定网站;化学;标签;

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