title: Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy.
abstract: Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity.
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Peuker
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Hanna
authors.initials: H
authors.lastname: Andersson
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Emil
authors.initials: E
authors.lastname: Gustavsson
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Kiran Sankar
authors.initials: KS
authors.lastname: Maiti
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Rafal
authors.initials: R
authors.lastname: Kania
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Alavi
authors.initials: A
authors.lastname: Karim
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Stephan
authors.initials: S
authors.lastname: Niebling
authors.affiliation: Swedish NMR Centre at the University of Gothenburg , P.O. Box 465, SE-405 30 Gothenburg, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Pedersen
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Mate
authors.initials: M
authors.lastname: Erdelyi
authors.affiliation: Department of Chemistry and Molecular Biology, University of Gothenburg , SE-405 30 Gothenburg, Sweden.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Westenhoff
mesh_terms: D002247:Carbon Isotopes; D049452:Green Fluorescent Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D010103:Oxygen Isotopes; D011789:Quantum Theory; D013055:Spectrophotometry, Infrared; D014443:Tyrosine / Q000737:chemistry; D014732:Vibration
chemical_list: D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D049452:Green Fluorescent Proteins; D014443:Tyrosine