Abstract
Studies of protein structure and function using single-molecule fluorescence resonance energy transfer (smFRET) benefit dramatically from the ability to site-specifically label proteins with small fluorescent dyes. Genetically encoding the unnatural amino acid (UAA) p-acetylphenylalanine is an efficient way to introduce commercially available fluorescent tags with high yield and specificity. This protocol describes the expression in Escherichia coli of proteins containing this UAA in response to the amber stop codon TAG. Proteins were purified with high yield and subsequently labeled with the hydroxylamine derivative of Alexa Fluor® 488 functioning as a fluorescent donor dye. The proteins were then labeled via maleimide coupling chemistry at a unique cysteine with the acceptor dye Alexa Fluor® 594 to yield a dual-labeled protein ready for subsequent smFRET observation.
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References
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Acknowledgments
I thank all members in the Deniz and Schultz laboratories at The Scripps Research Institute, especially Dr. Brustad for the good collaborations. The critical proofreading of this protocol by Dr. VanDelinder is also very much appreciated. Finally, I want to thank my laboratory members for their productive discussions, and the EMBL and the Emmy Noether Program of the DFG for funding my laboratory.
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Lemke, E.A. (2011). Site-Specific Labeling of Proteins for Single-Molecule FRET Measurements Using Genetically Encoded Ketone Functionalities. In: Mark, S. (eds) Bioconjugation Protocols. Methods in Molecular Biology, vol 751. Humana Press. https://doi.org/10.1007/978-1-61779-151-2_1
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DOI: https://doi.org/10.1007/978-1-61779-151-2_1
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