title: O-tert-Butyltyrosine, an NMR tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities.
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 concentrated protein solutions than usual, providing access to Kd values 1 order of magnitude lower than established NMR methods that employ direct protein detection for Kd measurements.
authors: †Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
authors: Wan-Na
authors: WN
authors: Chen
authors: †Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
authors: Kekini Vahini
authors: KV
authors: Kuppan
authors: ‡Monash Institute of Pharmaceutical Sciences, Parkville, VIC 3052, Australia.
authors: Michael David
authors: MD
authors: Lee
authors: §Latvian Institute of Organic Synthesis, Riga 1006, Latvia.
authors: Kristaps
authors: K
authors: Jaudzems
authors: †Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
authors: Thomas
authors: T
authors: Huber
authors: †Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia.
authors: Gottfried
authors: G
authors: Otting
mesh_terms: D053488:DnaB Helicases / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D007553:Isotope Labeling; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D008970:Molecular Weight; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C024096:3'-tert-butyltyrosine; D008024:Ligands; D014443:Tyrosine; D053488:DnaB Helicases