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Efficient Synthesis and In Vivo Incorporation of Acridon-2-ylalanine, a Fluorescent Amino Acid for Lifetime and FRET/LRET Studies

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  • The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in E. coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87% yield over five steps from Tyr, and the identification of an Acd synthetase by screening candidate enzymes previously evolved from M. janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Förster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu³⁺ to monitor protein folding and binding interactions.
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  • Speight, L. C., Muthusamy, A. K., Goldberg, J. M., Warner, J. B., Wissner, R. F., Willi, T. S., ... & Petersson, E. J. (2013). Efficient synthesis and in vivo incorporation of acridon-2-ylalanine, a fluorescent amino acid for lifetime and förster resonance energy transfer/luminescence resonance energy transfer studies. Journal of the American Chemical Society, 135(50), 18806-18814. doi:10.1021/ja403247j
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  • 135
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  • 50
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  • This work was supported by funding from the University of Pennsylvania, the Searle Scholars Program (10-SSP-214 to EJP), the National Institutes of Health (NIH NS081033 to EJP), the National Science Foundation (NSF MCB-0448297 to RAM), and the Cell Imaging and Analysis Facilities and Services Core of the EHSC, Oregon State University (P30 ES00210, NIEHS, NIH to RAM).
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