Monteagudo-Cascales Elizabet, Cano-Muñoz Mario, Genova Roberta, Cabrera Juan J, Matilla Miguel A, Krell Tino
Department of Biotechnology and Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, Granada 18008, Spain.
FEMS Microbiol Rev. 2025 Jan 14;49. doi: 10.1093/femsre/fuaf033.
Bacteria sense and respond to changing environmental conditions using a diverse range of receptors. Currently, the signals recognized by most receptors remain unknown, thereby limiting our understanding of their function. Since its introduction a decade ago, ligand screening by the thermal-shift assay has identified the signal molecules recognized by numerous receptors, solute-binding proteins, and transcriptional regulators. This progress is summarized in this review. Signal identification is facilitated by the fact that ligand-binding domains can be generated as individual soluble proteins that retain the signal-binding capabilities of the full-length proteins. Various issues relevant to the reliability of the thermal shift assay are discussed, including false-positive and false-negative results, the value of a protein pH screen prior to ligand screening, and the need to verify results with methods for the direct study of ligand binding, such as isothermal titration calorimetry. This review was inspired by the XVIII conference on Bacterial Locomotion and Signal Transduction (Cancun, January 2025), where several notable advances were reported based on the application of the thermal shift assay.
细菌利用多种受体感知并响应不断变化的环境条件。目前，大多数受体识别的信号仍不为人知，这限制了我们对其功能的理解。自十年前引入以来，通过热位移分析进行的配体筛选已鉴定出众多受体、溶质结合蛋白和转录调节因子所识别的信号分子。本综述总结了这一进展。配体结合结构域可以作为单独的可溶性蛋白产生，这些蛋白保留了全长蛋白的信号结合能力，这一事实有助于信号识别。讨论了与热位移分析可靠性相关的各种问题，包括假阳性和假阴性结果、配体筛选前蛋白质pH筛选的价值，以及需要用直接研究配体结合的方法（如等温滴定量热法）验证结果。本综述的灵感来自于第十八届细菌运动与信号转导会议（2025年1月，坎昆），会上报告了基于热位移分析应用的几项显著进展。
