On the acquisition and analysis of microscale thermophoresis data.
On the acquisition and analysis of microscale thermophoresis data.
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DOI：
10.1016/j.ab.2015.12.013
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发表时间：
2016-03-01
影响因子：
2.9
通讯作者：
Brautigam CA
中科院分区：
文献类型：
--
作者：
Scheuermann TH;Padrick SB;Gardner KH;Brautigam CA
对支撑细胞功能的分子机制的全面理解取决于对大分子结合的能量学的详细描述，大分子结合的能量学通常由平衡解离常数Kd来量化。虽然有许多生物物理方法可以用来获得KD，但本报告的重点是一种相对较新的方法，称为微尺度热导入(MST)。在MST实验中，用红外激光照射填充了含有染料标记溶质的溶液的毛细管，迅速产生温度梯度。分子将沿着这个梯度迁移，导致观察到的荧光发生变化。由于标记分子的净迁移将取决于它们的配体状态，因此可以根据MST数据构建作为配体浓度函数的结合曲线，并进行分析以确定Kd。在此，模拟演示了在当前仪器中可以测量的Kd的极限。他们还表明，当计划和执行MST实验时，结合动力学是一个主要考虑因素。此外，对两种蛋白质相互作用的研究说明了在获取和分析MST数据时遇到的挑战。结合起来，这些方法表明了一套执行和分析MST实验的最佳实践。还介绍了用于严格数据分析的软件。
A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, KD. While many biophysical methods may be used to obtain KD, the focus of this report is a relatively new method called “microscale thermophoresis” (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve can be constructed as a function of ligand concentration from MST data and analyzed to determine KD. Herein, simulations demonstrate the limits of KD that can be measured in current instrumentation. They also show that binding kinetics are a major concern when planning and executing MST experiments. Additionally, studies of two protein-protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced.