Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range. Furthermore, Mcm/Acd labeling can be combined with tryptophan fluorescence in three color FRET to monitor multiple interactions in one experiment.
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| http://dx.doi.org/10.1039/c7cc05492k | DOI Listing |
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