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

Abstract

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.

Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes

“…The spectral overlap between the fluorescence emission of oxazole 1a and the absorption spectrum of Acd is shown in Figure S4. The predicted R 0 value for these two fluorophores was calculated 25 to be 32.8 Å, while the distance between the α -carbon atoms of residues 39 and 66 is 17.0 Å based on X-ray crystallography (Figure 6B). By decoding the four-base codon CGGG with the acridon-2-ylalanyl-tRNA CCCG and the nonsense codon UAG with the oxazolyl-tRNA CUA , we incorporated Acd and compound 1a into GFP at positions 39 and 66, respectively.…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…1B42) and acridon-2-ylalanine ( Fig. 1B45) (Wang et al 2006a;Speight et al 2013). 7HC was inserted into bacterial tubulin FtsZ and used to track FtsZ localization during cell division (Charbon et al 2011).…”

Fluorescent CD4 probe for potential HIV-1 gp120 protein detection

“…[9][10][11] As a fluorescence acceptor, the amino acid acridon-2-ylalanine (Acd) is an attractive fluorophore because of its high quantum yield, high photostability and small size. 12 Its small size enables it to minimally perturb the folding of proteins in which it is incorporated. In this study, we prepared a fluorescent mD1 protein sensor that can specifically bind to the conserved domain of HIV gp120 protein.…”

“…12,14 The Acd absorption spectrum overlaps with the tail of the tryptophan (Trp) emission spectrum. Thus, it can form a FRET pair with Trp, with a Förster radius (R 0 ) of 23 Å.…”

“…12 However, in a steady state fluorescence Stern-Volmer titration assay, Trp and tyrosine (Tyr) can significantly quench the fluorescence of Acd through a photoinduced electron transfer (PET) mechanism when their distance is less than 15 Å. 12 These opposing effects of Trp on the fluorescence emission of Acd complicate the use of this fluorophore for the study protein conformation changes, but potentially may provide multiple mechanisms to monitor protein-protein interactions. Accordingly we began our study by exploring the ability of Trp and Acd to form a FRET pair in the mD1 protein.…”