Identification of Tunable, Environmentally Responsive Fluorogenic Dyes by High-Throughput Screening.

Small molecule dyes remain essential biological tools, yet only a handful of environmentally responsive fluorogenic small molecules are available for routine characterization of protein state. Here, we report the development and execution of a high throughput screen to identify compounds that increase in fluorescence in response to binding of lipophilic sites of proteins. This effort yielded two small molecules that potently indicate the presence of a range of common proteins and outperform common dyes in differential scanning fluorimetry experiments. Structure activity relationship studies revealed that these two scaffolds can be tuned both for their quantum yields and emission wavelengths. This work affords a straightforward framework for the discovery of new fluorophores and adds two fluorogenic probes to the toolbox for studying protein state.