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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
Links dx.doi.org/10.1021/jacs.5b12680
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

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.

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