A green-fluorogenic, cysteine-reactive dye enables thermal shift-based high-throughput screening with membrane proteins

Thermal shift assays are useful tools in the biophysical screening of protein targets. They require little assay establishment and no tool compounds, making them particularly useful for proteins with no known natural ligands. Their use in compound-screening is typically limited to soluble proteins, however, as membrane proteins that are solubilized in detergent cannot be routinely used with the fluorogenic dyes that detect hydrophobic surfaces upon protein unfolding. As an alternative, a method has been described that utilizes the frequent occurrence of cysteines within the transmembrane region of membrane proteins in combination with fluorogenic, cysteine-reactive dyes to record melting curves of membrane proteins. Here, we evaluate several novel candidate dyes and find that the dye Naph-EA-Mal can reliably record melting points of membrane proteins at low concentrations and with high tolerance for different buffers or classes of membrane proteins. We apply the thermal shift assay with the new dye to a buffer optimization for the ion channel GIRK4 and to a fragment screen with the solute carrier Spns2. Thereby, we discover a new binder scaffold that may be useful in the development of Spns2 inhibitors in the future.