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Issue 80, 2017
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Multicolor protein FRET with tryptophan, selective coumarin-cysteine labeling, and genetic acridonylalanine encoding

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Abstract

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.

Graphical abstract: Multicolor protein FRET with tryptophan, selective coumarin-cysteine labeling, and genetic acridonylalanine encoding

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Publication details

The article was received on 15 Jul 2017, accepted on 20 Sep 2017 and first published on 26 Sep 2017


Article type: Communication
DOI: 10.1039/C7CC05492K
Chem. Commun., 2017,53, 11072-11075

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    Multicolor protein FRET with tryptophan, selective coumarin-cysteine labeling, and genetic acridonylalanine encoding

    J. J. Ferrie, N. Ieda, C. M. Haney, C. R. Walters, I. Sungwienwong, J. Yoon and E. J. Petersson, Chem. Commun., 2017, 53, 11072
    DOI: 10.1039/C7CC05492K

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