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Cellular Structural Biology (IBI-6)
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Cotranslational Incorporation into Proteins of a Fluorophore for smFRET

Single-molecule FRET (smFRET) is a powerful tool to investigate conformational changes of biological molecules. In general, smFRET studies require protein samples that are site-specifically double-labeled with a pair of donor and acceptor fluorophores. The common approaches to produce such samples cannot be applied when studying the synthesis and folding of the polypeptide chain on the ribosome. The best strategy is to incorporate two fluorescent amino acids co-translationally using cell-free protein synthesis systems. Here, we demonstrate the co-translational site-specific incorporation into a model protein of Atto633, a dye with excellent photophysical properties, suitable for single molecule spectroscopy, together with a second dye using a combination of the sense cysteine and the nonsense amber codon. In this work we show that co-translational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies.

(Sadoine et al., ACS Synth. Biol. 2018, 7, 405-411; doi: 10.1021/acssynbio.7b00433)