MicroScale Thermophoresis: Interaction analysis and beyond
MicroScale Thermophoresis: Interaction analysis and beyond
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DOI：
10.1016/j.molstruc.2014.03.009
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发表时间：
2014-12-05
影响因子：
3.8
通讯作者：
Breitsprecher, Dennis
中科院分区：
文献类型：
--
作者：
Jerabek-Willemsen, Moran;Andre, Timon;Breitsprecher, Dennis
微尺度热泳（MST）是一种强大的技术来量化生物分子的相互作用。它是基于热泳，分子在温度梯度中的定向运动，这强烈取决于各种分子性质，如大小，电荷，水合壳或构象。因此，该技术对几乎任何分子性质的变化都非常敏感，可以精确定量分子事件，而不受所研究样品的大小或性质的影响。在MST实验中，红外激光会诱导温度梯度。分子通过温度梯度的定向运动使用共价连接的或内在的荧光团进行检测和定量。MST结合了荧光检测的精确性和热泳的可变性和灵敏性，为研究分子间相互作用提供了一种灵活、稳健和快速的方法。本文综述了MST技术的最新进展和发展，重点介绍了MST在标准生物分子相互作用研究之外的应用。通过使用不同的模型系统，我们介绍了替代MST应用-如结合化学计量和结合模式的测定，蛋白质展开，热力学和酶动力学的分析。此外，我们证明了MST的能力，以量化高亲和力的相互作用与解离常数（K（d）s）在低皮摩尔（pM）范围内，以及蛋白质-蛋白质的相互作用在纯哺乳动物细胞裂解物。(C)2014作者由爱思唯尔公司出版
MicroScale Thermophoresis (MST) is a powerful technique to quantify biomolecular interactions. It is based on thermophoresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell or conformation. Thus, this technique is highly sensitive to virtually any change in molecular properties, allowing for a precise quantification of molecular events independent of the size or nature of the investigated specimen.During a MST experiment, a temperature gradient is induced by an infrared laser. The directed movement of molecules through the temperature gradient is detected and quantified using either covalently attached or intrinsic fluorophores. By combining the precision of fluorescence detection with the variability and sensitivity of thermophoresis, MST provides a flexible, robust and fast way to dissect molecular interactions.In this review, we present recent progress and developments in MST technology and focus on MST applications beyond standard biomolecular interaction studies. By using different model systems, we introduce alternative MST applications - such as determination of binding stoichiometries and binding modes, analysis of protein unfolding, thermodynamics and enzyme kinetics. In addition, wedemonstrate the capability of MST to quantify high-affinity interactions with dissociation constants (K(d)s) in the low picomolar (pM) range as well as protein-protein interactions in pure mammalian cell lysates. (C) 2014 The Authors. Published by Elsevier B.V.