Jagilinki Bhanu P, Willis Mark A, Mus Florence, Sharma Ritika, Pellows Lauren M, Mulder David W, Yang Zhi-Yong, Seefeldt Lance C, King Paul W, Dukovic Gordana, Peters John W
Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, OK, USA.
Institute of Biological Chemistry, Washington State University, Pullman, WA, USA.
Bio Protoc. 2024 Aug 5;14(15):e5041. doi: 10.21769/BioProtoc.5041.
Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a -based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this , we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein-protein interactions, protein-ligand interactions, and protein-nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems. Key features • Established procedures for loading and sealing tubes in an anaerobic chamber for subsequent analysis. • Sodium dithionite (NaDT) could easily be substituted with one electron-reduced 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium [(SPr)V•] to perform sensitive biophysical assays on oxygen-sensitive proteins like the MoFe protein. • Established MST as an experimental tool to quantify binding affinities in novel enzyme-quantum dot biohybrid complexes that are extremely oxygen-sensitive.
微量热泳技术（MST）是一种用于测量分子相互作用强度的技术。MST是一种基于[具体原理未提及]的技术，它通过监测红外激光触发的温度梯度下荧光标记分子移动所伴随的荧光变化。与其他检测分子相互作用的方法相比，如等温滴定量热法、核磁共振、生物膜干涉术和表面等离子体共振，MST具有优势，它所需样品量小，无需固定，且具有高灵敏度荧光检测。此外，由于该方法涉及将样品加载到易于密封的毛细管中，因此可适用于分析对氧气敏感的样品。在本文中，我们描述了为使MST能够用于检测蛋白质-蛋白质相互作用、蛋白质-配体相互作用和蛋白质-纳米晶体相互作用而进行的故障排除和优化工作。所开发程序中的关键要素包括：1）在厌氧箱中进行加载和密封的能力，以便使用置于空气中工作台上的NanoTemper MST进行分析；2）确定与数据采集兼容的最佳还原剂，并有效防止痕量氧气干扰；3）优化数据采集和分析程序。这些程序为在这些技术要求较高的系统中确定分子相互作用的决定因素奠定了基础。关键特性 • 建立了在厌氧箱中加载和密封试管以便后续分析的程序。 • 连二亚硫酸钠（NaDT）可轻松被单电子还原的1,1'-双（3-磺丙基）-4,4'-联吡啶鎓[（SPr）V•]替代，以对诸如钼铁蛋白等对氧气敏感的蛋白质进行灵敏的生物物理分析。 • 确立了MST作为一种实验工具，用于量化新型酶-量子点生物杂交复合物中的结合亲和力，该复合物对氧气极其敏感。
