Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy

Journal of the American Chemical Society
Sebastian PeukerSebastian Westenhoff

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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Citations

Jan 7, 2017·Chemical Reviews·Ayanjeet GhoshMartin T Zanni
Jul 10, 2018·Journal of Breath Research·Kiran Sankar MaitiAlexander Apolonski
Nov 8, 2019·Chembiochem : a European Journal of Chemical Biology·Annika UrbanekPau Bernadó
Jun 17, 2020·Bioscience, Biotechnology, and Biochemistry·Kakeru SuzukiYuzuru Tozawa
Aug 15, 2020·Frontiers in Bioengineering and Biotechnology·Yang WuHao Qi
Mar 24, 2020·Chemical Reviews·Victor A Lorenz-Fonfria
Apr 9, 2020·Journal of the American Chemical Society·Annika UrbanekPau Bernadó
Sep 26, 2017·New Biotechnology·Daniel FoshagFrank Bernhard
Jul 28, 2021·Annual Review of Analytical Chemistry·Goran W TumbicMegan C Thielges

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