Cotranslational Incorporation into Proteins of a Fluorophore Suitable for smFRET Studies
| Autor: | Michele Cerminara, Alexandros Katranidis, Joerg Fitter, Mayuri Sadoine, Michael Gerrits |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Fluorophore Biomedical Engineering 010402 general chemistry Protein Engineering 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) Ribosome 03 medical and health sciences chemistry.chemical_compound Calmodulin Protein biosynthesis Escherichia coli Fluorescence Resonance Energy Transfer Humans Fluorescent Dyes chemistry.chemical_classification Staining and Labeling Biomolecule General Medicine Fluorescence 0104 chemical sciences Amino acid Folding (chemistry) 030104 developmental biology Förster resonance energy transfer chemistry Biophysics Protein Modification Translational |
| Zdroj: | ACS synthetic biology. 7(2) |
| ISSN: | 2161-5063 |
| Popis: | 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 cotranslationally using cell-free protein synthesis systems. Here, we demonstrate the cotranslational 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 cotranslational incorporation of good fluorophores into proteins is a viable strategy to produce suitable samples for smFRET studies. |
| Databáze: | OpenAIRE |
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