Racemic phosphorothioate as a tool for NMR investigations of protein-DNA complexes.
Racemic phosphorothioate as a tool for NMR investigations of protein-DNA complexes.
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DOI：
10.1007/s10858-020-00333-x
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发表时间：
2020-09
影响因子：
2.7
通讯作者：
Iwahara J
中科院分区：
文献类型：
--
作者：
Nepravishta R;Pletka CC;Iwahara J
蛋白质-核酸缔合的主要驱动力是通过带正电荷的碱性侧链和带负电荷的磷酸盐的离子对的静电相互作用。为了更好地理解蛋白质如何扫描DNA并识别特定的签名，重要的是要获得对蛋白质-DNA界面处基本侧链行为的原子水平的洞察。NMR光谱是研究蛋白质-DNA相互作用的结构、动力学和动力学方面的有力工具。然而，在分子界面处的基本侧链阳离子部分的共振分配仍然是一个重大挑战。在这里，我们提出了一种快速，稳健，廉价的方法，极大地促进了界面部分的共振分配，也允许两个DNA双链体之间的蛋白质易位的动力学测量。该方法利用外消旋硫代磷酸酯在与蛋白质侧链相互作用的磷酸酯的位置处的位点特异性掺入。这种修饰保留了磷酸盐的电荷，因此是温和的，但会导致近端蛋白质侧链的显着化学位移扰动，这有利于共振分配。由于修饰的外消旋性质，对于具有并入的硫代磷酸酯基团的不同非对映异构体RP和SP的物质，观察到两种不同的化学位移。RP和SP DNA双链体之间的蛋白质分子交换的动力学信息可以通过15 Nz交换光谱获得。我们展示了这种方法的应用程序的napedia同源域-DNA复合物和CREB 1碱性亮氨酸拉链（bZIP）-DNA复合物。
A major driving force for protein-nucleic acid association is electrostatic interactions via ion pairs of the positively charged basic side chains and negatively charged phosphates. For a better understanding of how proteins scan DNA and recognize particular signatures, it is important to gain atomic-level insight into the behavior of basic side chains at the protein-DNA interfaces. NMR spectroscopy is a powerful tool for investigating the structural, dynamic, and kinetic aspects of protein-DNA interactions. However, resonance assignment of basic side-chain cationic moieties at the molecular interfaces remains to be a major challenge. Here, we propose a fast, robust, and inexpensive approach that greatly facilitates resonance assignment of interfacial moieties and also allows for kinetic measurements of protein translocation between two DNA duplexes. This approach utilizes site-specific incorporation of racemic phosphorothioate at the position of a phosphate that interacts with a protein side chain. This modification retains the electric charge of phosphate and therefore is mild, but causes significant chemical shift perturbations for the proximal protein side chains, which facilitates resonance assignment. Due to the racemic nature of the modification, two different chemical shifts are observed for the species with different diastereomers RP and SP of the incorporated phosphorothioate group. Kinetic information on the exchange of the protein molecule between RP and SP DNA duplexes can be obtained by 15Nz exchange spectroscopy. We demonstrate the applications of this approach to the Antennapedia homeodomain–DNA complex and the CREB1 basic leucine-zipper (bZIP)–DNA complex.
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影响因子：
14.9
作者：
Jones, S;Daley, DTA;Thornton, JM
通讯作者：
Thornton, JM
影响因子：
2.9
作者：
Dan Nguyen;Hoffpauir, Zoe A.;Iwahara, Junji
通讯作者：
Iwahara, Junji
DOI：
10.1016/j.ymeth.2018.05.004
发表时间：
2018-09-15
期刊：
Methods (San Diego, Calif.)
影响因子：
--
作者：
Iwahara J;Zandarashvili L;Kemme CA;Esadze A
通讯作者：
Esadze A
影响因子：
15
作者：
Andre, Ingemar;Linse, Sara;Mulder, Frans A. A.
通讯作者：
Mulder, Frans A. A.
影响因子：
2.7
作者：
DELAGLIO, F;GRZESIEK, S;BAX, A
通讯作者：
BAX, A