A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation.

ABSTRACT: 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 ?-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 ?-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.

SUBMITTER: Johnson OT 

PROVIDER: S-EPMC6482830 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature