Fluorescence anisotropy-based salt-titration approach to characterize protein-nucleic acid interactions.
Fluorescence anisotropy-based salt-titration approach to characterize protein-nucleic acid interactions.
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DOI：
10.1007/978-1-4939-2214-7_23
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Musier-Forsyth, Karin
中科院分区：
文献类型：
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作者：
Rye-McCurdy, Tiffiny;Rouzina, Ioulia;Musier-Forsyth, Karin
许多蛋白质通过与RNA或DNA的阴离子磷酸骨架静电相互作用的阳离子残基结合核酸（NA）。这些静电相互作用通常对NA序列和结构不敏感，但赋予结合相互作用强的盐依赖性。相反，盐独立的非静电接触反映了更特异的结合相互作用。具有通过柔性接头连接的多个阳离子NA结合结构域的蛋白质，例如HIV-1 Gag多蛋白，可以以不同的方式结合不同的NA分子。例如，Gag与HIV-1 RNA基因组的Psi包装信号的结合优化了这种蛋白质-RNA相互作用的特异性非静电结合组分。相反，Gag与非psi RNA的结合以特定接触为代价优化了静电相互作用。在这里，我们描述了一种基于荧光各向异性的盐滴定方法，该方法允许在单一测定中以定量方式对任何蛋白质-NA复合物的静电和非静电结合组分进行完整表征。
Many proteins bind nucleic acids (NA) via cationic residues that interact electrostatically with the anionic phosphate backbone of RNA or DNA. These electrostatic interactions are often insensitive to NA sequence and structure, but confer strong salt dependence to the binding interactions. In contrast, salt-independent non-electrostatic contacts reflect more specific binding interactions. Proteins with multiple cationic NA-binding domains connected by flexible linkers, such as the HIV-1 Gag polyprotein, may bind different NA molecules in distinct ways. For example, Gag binding to the Psi-packaging signal of the HIV-1 RNA genome optimizes the specific non-electrostatic binding component of this protein-RNA interaction. In contrast, Gag binding to a non-psi RNA optimizes the electrostatic interactions at the expense of specific contacts. Here, we describe a fluorescence anisotropy-based salt-titration approach that allows complete characterization of both electrostatic and non-electrostatic binding components for any protein-NA complex in a quantitative manner within a single assay.