SSB-DNA binding monitored by fluorescence intensity and anisotropy.
SSB-DNA binding monitored by fluorescence intensity and anisotropy.
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DOI：
10.1007/978-1-62703-032-8_4
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Lohman, Timothy M
中科院分区：
文献类型：
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作者：
Kozlov, Alexander G;Galletto, Roberto;Lohman, Timothy M
荧光方法已被证明是非常有用的工具，定量研究的平衡和动力学的蛋白质-DNA相互作用。如果蛋白质含有色氨酸（Trp），通常情况下，并且蛋白质的内在Trp荧光发生变化，则可以使用这种信号变化（猝灭/增强）来监测结合。人们还可以将外源荧光团连接到蛋白质或DNA上，并监测由于荧光强度变化或荧光各向异性变化而引起的结合。这样的平衡研究可以提供重要的定量信息的化学计量（封闭的网站大小，结合位点的数量）和相互作用的能量（亲和力和协同性）。这些信息是理解蛋白质-DNA相互作用机制所必需的。对于具有非统一化学计量的系统（例如，结合多个配体的蛋白质）是荧光信号（强度或各向异性）的变化与平均结合程度之间的关系的知识。在这里，我们描述的程序，使用荧光的方法来检查大肠杆菌单链DNA结合蛋白（SSB）和耐辐射异常球菌SSB的化学计量和平衡结合亲和力与长的聚合物ssDNA，以确定一个闭塞的网站大小。我们还提供了SSB与较短寡核苷酸结合的研究实例，以证明数据分析和拟合到适当的模型（监测荧光强度或各向异性），以获得平衡结合参数的定量估计。我们强调，溶液条件（特别是盐浓度和类型）不仅会影响结合亲和力，而且会影响SSB寡聚体结合ssDNA的模式。
Fluorescence methods have proven to be extremely useful tools for quantitative studies of the equilibria and kinetics of protein-DNA interactions. If the protein contains tryptophan (Trp), as is often the case, and there is a change in intrinsic Trp fluorescence of the protein, one can use this change in signal (quenching/enhancement) to monitor binding. One can also attach an extrinsic fluorophore to either the protein or the DNA and monitor binding due to a change in fluorescence intensity or a change in fluorescence anisotropy. Such equilibrium studies can provide important quantitative information on stoichiometries (occluded site size, number of binding sites) and energetics (affinities and cooperativities) of the interactions. This information is needed to understand the mechanisms of protein-DNA interactions. A critical aspect of such approaches for systems that have non-unity stoichiometries (e.g., a protein that binds multiple ligands) is knowledge of the relationship between the change in fluorescence signal (intensity or anisotropy) and the average extent of binding. Here we describe procedures for using fluorescence approaches to examine the stoichiometries and equilibrium binding affinities of Escherichia coli single-stranded DNA-binding protein (SSB) and Deinococcus radiodurans SSB with long polymeric ssDNA to determine an occluded site size. We also provide examples of studies of SSB binding to shorter oligonucleotides to demonstrate analysis and fitting of the data to an appropriate model (monitoring fluorescence intensity or anisotropy) to obtain quantitative estimates of equilibrium binding parameters. We emphasize that the solution conditions (especially salt concentration and type) can influence not only the binding affinity, but also the mode by which an SSB oligomer binds ssDNA.