O-tert-Butyltyrosine, an NMR tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities

Journal of the American Chemical Society
Wan-Na ChenGottfried Otting

Abstract

O-tert-Butyltyrosine (Tby) is an unnatural amino acid that can be site-specifically incorporated into proteins using established orthogonal aminoacyl-tRNA synthetase/tRNA systems. Here we show that the tert-butyl group presents an outstanding NMR tag that can readily be observed in one-dimensional (1)H NMR spectra without any isotope labeling. Owing to rapid bond rotations and the chemical equivalence of the protons of a solvent-exposed tert-butyl group from Tby, the singlet resonance from the tert-butyl group generates an easily detectable narrow signal in a spectral region with limited overlap with other methyl resonances. The potential of the tert-butyl (1)H NMR signal in protein research is illustrated by the observation and assignment of two resonances in the Bacillus stearothermophilus DnaB hexamer (320 kDa), demonstrating that this protein preferentially assumes a 3-fold rather than 6-fold symmetry in solution, and by the quantitative measurement of the submicromolar dissociation constant Kd (0.2 μM) of the complex between glutamate and the Escherichia coli aspartate/glutamate binding protein (DEBP, 32 kDa). The outstanding signal height of the (1)H NMR signal of the Tby tert-butyl group allows Kd measurements using less...Continue Reading

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Citations

Mar 15, 2016·Journal of the American Chemical Society·Wan-Na ChenGottfried Otting
Jun 20, 2016·Current Opinion in Structural Biology·Petra Neumann-Staubitz, Heinz Neumann
Mar 23, 2018·Journal of Biomolecular NMR·Walter BeckerChristoph Nitsche
Dec 14, 2017·Quarterly Reviews of Biophysics·Galia T Debelouchina, Tom W Muir
Oct 15, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Abdul-Hamid EmwasMariusz Jaremko
Feb 20, 2018·ACS Medicinal Chemistry Letters·Wan-Na Chen, Gottfried Otting
Sep 11, 2021·Journal of Biomolecular NMR·Rashmi VoletiJosep Rizo

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