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

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.

SUBMITTER: Ferrie JJ

PROVIDER: S-EPMC6533536 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Ferrie John J JJ Ieda Naoya N Haney Conor M CM Walters Christopher R CR Sungwienwong Itthipol I Yoon Jimin J Petersson E James EJ

Chemical communications (Cambridge, England) 20171001 80

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 mi ...[more]

PMID: 28948265

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