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Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy

Journal article
Authors Sebastian Peuker
Hanna Andersson
Emil Gustavsson
K. S. Maiti
R Kania
Alavi Karim
Stephan Niebling
Anders Pedersen
Mate Erdelyi
Sebastian Westenhoff
Published in Journal of the American Chemical Society
Volume 138
Issue 7
Pages 2312-2318
ISSN 0002-7863
Publication year 2016
Published at Swedish NMR Centre at Göteborg University
Department of Chemistry and Molecular Biology
Pages 2312-2318
Language en
Keywords green fluorescent protein, 2-dimensional infrared-spectroscopy, unnatural amino-acids, proton-transfer, chemical aminoacylation, transfer-rnas, genetic-code, active-site, dynamics, expression, Chemistry
Subject categories Biochemistry and Molecular Biology, Chemical Sciences


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.

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