Determination of protein-ligand interactions using differential scanning fluorimetry.
Determination of protein-ligand interactions using differential scanning fluorimetry.
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DOI：
10.3791/51809
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发表时间：
2014-09-13
期刊：
影响因子：
--
通讯作者：
Harmer NJ
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文献类型：
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作者：
Vivoli M;Novak HR;Littlechild JA;Harmer NJ
目前有多种方法可用于确定蛋白质和相互作用的小分子之间的解离常数。然而，其中大多数需要使用专业设备，并且通常需要一定程度的专业知识才能有效地建立可靠的实验和分析数据。差示扫描荧光测定法 (DSF) 越来越多地被用作一种稳健的方法，用于初步筛选相互作用的小分子蛋白质，用于识别生理伙伴或发现命中。该技术的优点是只需要一台适合定量PCR的PCR仪，因此大多数机构都有合适的仪器；已有一系列优秀的协议可供使用；文献中有多次使用该方法的先例。过去的工作提出了几种从 DSF 数据计算解离常数的方法，但这些方法在数学上要求很高。在这里，我们演示了一种根据适量 DSF 实验数据估计解离常数的方法。这些数据通常可以在一天内收集和分析。我们演示了如何使用不同的模型来拟合从简单结合事件收集的数据，以及存在协作结合或独立结合位点的位置。最后，我们提供了一个在标准模型不适用的情况下进行数据分析的示例。这些方法通过从市售对照蛋白和我们研究项目中的两种蛋白收集的数据来说明。总体而言，我们的方法为研究人员提供了一种使用 DSF 快速进一步了解蛋白质-配体相互作用的简单方法。
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.