Department of Physiology, University of Texas Southwestern Medical Center.
Department of Physiology, University of Texas Southwestern Medical Center; Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center;
J Vis Exp. 2024 Aug 9(210). doi: 10.3791/67139.
Defining the biological importance of proteins with unknown functions poses a significant obstacle in understanding cellular processes. Although bioinformatic and structural predictions have contributed to the study of unknown proteins, in vitro experimental validations are often hampered by the optimal conditions and cofactors required for biochemical activity. Cofactor binding is not only essential for the activity of some enzymes but may also enhance the thermal stability of the protein. One practical application of this phenomenon lies in utilizing the change in thermal stability, as measured by alterations in the protein's melting temperature, to probe ligand binding. Thermal shift assay (TSA) can be used to analyze the binding of different ligands to the protein of interest or find a stabilizing condition to perform experiments such as X-ray crystallography. Here we will describe a protocol for TSA utilizing the pseudokinase, Selenoprotein O (SelO), for a simple and high-throughput method for testing metal and nucleotide binding. In contrast to canonical kinases, SelO binds ATP in an inverted orientation to catalyze the transfer of AMP to the hydroxyl side chains of proteins in a posttranslational modification known as protein AMPylation. By leveraging the shift in the melting temperatures, we provide crucial insights into the molecular interactions underlying SelO function.
确定具有未知功能的蛋白质的生物学重要性是理解细胞过程的一个重大障碍。尽管生物信息学和结构预测有助于研究未知蛋白质，但体外实验验证常常受到生化活性所需的最佳条件和辅因子的限制。辅因子结合不仅是某些酶活性所必需的，而且还可以增强蛋白质的热稳定性。这种现象的一个实际应用在于利用热稳定性的变化，即通过改变蛋白质的熔点来探测配体结合。热位移分析（TSA）可用于分析不同配体与目标蛋白质的结合，或找到稳定条件来进行 X 射线晶体学等实验。在这里，我们将描述一种利用假激酶硒蛋白 O（SelO）进行 TSA 的方案，这是一种简单、高通量的方法，可用于测试金属和核苷酸结合。与典型的激酶不同，SelO 以倒置的方式结合 ATP，以催化 AMP 向蛋白质的羟基侧链转移，这种翻译后修饰被称为蛋白质 AMP 化。通过利用熔点的变化，我们深入了解了 SelO 功能的分子相互作用。
