High-Throughput Screening to Identify Chemoreceptor Ligands
The majority of bacterial chemoreceptors remain functionally un-annotated. The knowledge of chemoreceptor function, however, is indispensable to understanding the evolution of the chemotaxis system in bacteria with different lifestyles. Significant progress in the annotation of chemoreceptor function has been made using experimental strategies that are based on the individual, genetically engineered ligand binding domain (LBD) of chemoreceptors. There is now evidence that all major classes of LBDs can be produced as individual domains that retain their ligand binding activity. Here, we provide a protocol for the combined use of high-throughput ligand screening using Differential Scanning Fluorimetry followed by Isothermal Titration Calorimetry to identify and characterize ligands that bind to recombinant chemoreceptor LBDs. This approach has been shown to be very efficient for determining the function of novel chemoreceptors.
KeywordsChemoreceptor Ligand binding domain Thermal-shift assays Isothermal titration calorimetry Differential scanning fluorimetry
We acknowledge financial support from FEDER funds and Fondo Social Europeo through grants from the Junta de Andalucía (grant CVI-7335) and the Spanish Ministry for Economy and Competitiveness (grants BIO2013-42297 and BIO2016-76779-P).