title: 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 Escherichia 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 Methanococcus 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 Eu(3+) to monitor protein folding and binding interactions.
authors.affiliation: University of Pennsylvania , Department of Chemistry, 231 South 34th Street, Philadelphia, Pennsylvania, 19104-6323, United States.
authors.forename: Lee C
authors.initials: LC
authors.lastname: Speight
authors.forename: Anand K
authors.initials: AK
authors.lastname: Muthusamy
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Goldberg
authors.forename: John B
authors.initials: JB
authors.lastname: Warner
authors.forename: Rebecca F
authors.initials: RF
authors.lastname: Wissner
authors.forename: Taylor S
authors.initials: TS
authors.lastname: Willi
authors.forename: Bradley F
authors.initials: BF
authors.lastname: Woodman
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.forename: E James
authors.initials: EJ
authors.lastname: Petersson
mesh_terms: D000166:Acridines / Q000138:chemical synthesis / Q000737:chemistry*; D000409:Alanine / Q000138:chemical synthesis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049449:Luminescence; D008958:Models, Molecular
chemical_list: D000166:Acridines; D000409:Alanine