Generation of intramolecular FRET probes via noncanonical amino acid mutagenesis

Abstract

Förster resonance energy transfer (FRET) probes are powerful tools to monitor protein–protein interactions and enzyme activities in a spatiotemporal manner in live cells. Using a combination of noncanonical amino acid (ncAA) mutagenesis and bioorthogonal labeling, we have developed intramolecular FRET probes consisting of a fluorescent protein and an organic dye within an individual protein. Herein we present a general approach to establish intramolecular FRET probes for imaging of protein activity in live cells.