Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy.

Peuker S, Andersson H, Gustavsson E, Maiti KS, Kania R, Karim A, Niebling S, Pedersen A, Erdelyi M, Westenhoff S

J. Am. Chem. Soc. 138 (7) 2312-2318 [2016-02-24; online 2016-02-10]

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.

Swedish NMR Centre (SNC) [Collaborative]

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PubMed 26796542

DOI 10.1021/jacs.5b12680

Crossref 10.1021/jacs.5b12680