Thermal shift assay to identify ligands for bacterial sensor proteins

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.