title: Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore.
abstract: A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid l-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifically incorporated into the recombinant enhanced cyan fluorescent protein (eCFP) at a permissible surface position ∼20 Å away from the protein fluorophore using amber suppression in Escherichia coli with an engineered cognate Methanococcus jannaschii tRNA synthetase. The resulting eCFP(Cou) exhibited almost quantitative intramolecular Förster resonance energy transfer (FRET) between its two fluorophores, showing brilliant cyan emission at 476 nm upon excitation in the near-UV at 365 nm (a wavelength easily accessible via conventional laboratory UV sources), in contrast to its natural counterpart. Thus, this fluorescent protein with unprecedented spectroscopic properties reveals an extreme apparent Stokes shift of ∼110 nm between the absorption wavelength of the coumaryl group and the emission wavelength of eCFP.
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85350 Freising-Weihenstephan, Germany.
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Kuhn
authors.forename: Marina
authors.initials: M
authors.lastname: Rubini
authors.forename: Michael A
authors.initials: MA
authors.lastname: Müller
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D017017:Methanococcus / Q000201:enzymology; D008958:Models, Molecular; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D000604:Amino Acyl-tRNA Synthetases