Multiplexed electrochemistry of DNA-bound metalloproteins.
Multiplexed electrochemistry of DNA-bound metalloproteins.
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DOI：
10.1021/ja4041779
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发表时间：
2013-08-14
影响因子：
15
通讯作者：
Barton, Jacqueline K.
中科院分区：
文献类型：
--
作者：
Pheeney, Catrina G.;Arnold, Anna R.;Grodick, Michael A.;Barton, Jacqueline K.
在这里，我们描述了含有 [4Fe-4S] 簇的 DNA 结合蛋白的多重电化学表征。 DNA 修饰电极已成为表征含有氧化还原辅因子的 DNA 修复蛋白的氧化还原化学的重要工具，而多重分析提供了一种并行探测不同复杂样品和底物以阐明这种化学的方法。核酸内切酶 III (EndoIII) 是一种含有 [4Fe-4S] 簇的 DNA 修复蛋白，可通过 DNA 介导的电荷传输进行多重分析，结果显示电化学行为与 DNA 形态的函数存在细微差异。在紧密和松散堆积的 DNA 薄膜上，EndoIII 的 DNA 结合还原的峰分裂、信号宽度、对 π 堆栈扰动的敏感性和动力学都得到了表征。 DNA 结合的 EndoIII 有两种不同的电子转移途径进行还原，要么通过 DNA 碱基堆叠，要么通过直接表面还原；紧密堆积的 DNA 薄膜，其中蛋白质的表面可及性有限，产生反映 DNA 介导的电子转移的电化学信号。此外，多重分析还可以比较 EndoIII 突变体的电化学，包括改变 [4Fe-4S] 簇周围静电的新突变家族。虽然该突变体家族的中点电位几乎没有变化，但 DNA 介导的电子转移效率的显着变化是显而易见的。基于这些蛋白质的稳定性，通过圆二色性检查，我们提出，电子转移途径不仅可以通过芳香残基的去除来扰动，还可以通过簇附近溶剂化的变化来扰动。
Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.
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DOI：
10.1126/science.1165921
发表时间：
2009-01-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Morrow TJ;Li M;Kim J;Mayer TS;Keating CD
通讯作者：
Keating CD
影响因子：
56.9
作者：
MURPHY, CJ;ARKIN, MR;BARTON, JK
通讯作者：
BARTON, JK
DOI：
10.1073/pnas.0906429106
发表时间：
2009-08-11
影响因子：
11.1
作者：
Lee, Paul E.;Demple, Bruce;Barton, Jacqueline K.
通讯作者：
Barton, Jacqueline K.
影响因子：
2.9
作者：
CUNNINGHAM, RP;ASAHARA, H;EMPTAGE, MH
通讯作者：
EMPTAGE, MH
影响因子：
15
作者：
Bonham, Andrew J.;Hsieh, Kuangwen;Ferguson, B. Scott;Vallee-Belisle, Alexis;Ricci, Francesco;Soh, H. Tom;Plaxco, Kevin W.
通讯作者：
Plaxco, Kevin W.