Protein labeling for FRET with methoxycoumarin and acridonylalanine

Site-specific protein labeling can be used to monitor protein motions and interactions in real time using Förster resonance energy transfer (FRET). While there are many fluorophores available for protein labeling, few FRET pairs are suitable for monitoring intramolecular protein motions without being disruptive to protein folding and function. Here, we describe the synthesis and use of a minimally perturbing FRET pair comprised of methoxycoumarin maleimide (Mcm-Mal) and acridonylalanine (Acd). Acd can be incorporated into a protein through unnatural amino acid mutagenesis. Mcm-Mal is fluorogenic when reacted with cysteine and can label cysteine/Acd double mutant proteins. This labeling strategy provides an easy to install FRET pair with a working range or 15-40Å, making it ideal for monitoring most intramolecular motions. Additionally, Mcm/Acd FRET can be combined with tryptophan fluorescence for monitoring multiple protein motions via three color FRET.