LAUSR

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E‐BP1 is an example of an intrinsically disordered protein that forms an α‐helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding‐induced structural changes are low‐throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E‐BP1 peptide that reports structural changes of its helix in high‐throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α‐helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high‐throughput screening to discover ligands that alter peptide secondary structure.