Novel Approaches in Selective Tryptophan Isotope Labeling by Using Escherichia coli Overexpression Media

Abstract

NMR-based investigations of large protein complexes require optimized isotopic labeling schemes. We report new methods to introduce stable isotopes into tryptophan residues; these are fine-tuned to the requirements of the particular protein NMR experiment. Selective backbone labeling was performed by using a new α-ketoacid precursor as an additive in cell-based overexpression media. Additionally, we developed synthetic routes to certain isotopologues of indole with (13)C-(1)H spin systems surrounded by (12)C and (2)H. The corresponding proteins, overexpressed in the presence of these precursor compounds, can be effectively analyzed for conformational changes in tryptophan residues in response to external stimuli, such as interaction with other proteins or small molecules.

Site-selective 13C labeling of proteins using erythrose

“…Pyruvate (Milbradt et al 2015), 4- 13 C erythrose in combination with deuterated pyruvate (Kasinath et al 2013), and more advanced chemically synthesized precursors for labeling of Trp (Schörghuber et al 2015), Tyr and Phe (Lichtenecker et al 2013), including perdeuteration of all other hydrogen positions in the aromatic side-chain. All these methods are common in-vivo labeling strategies using E. coli for protein expression.…”

Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water

“…[9] These limitations in the study of IDPs can be overcome by using hyperpolarized HDO [10] produced through dissolution dynamic nuclear polarization (D-DNP). [12] Such sparse spectra are reminiscent of "spectral dilution" techniques that rely on 13 Cl abelling of selected amino acids. [12] Such sparse spectra are reminiscent of "spectral dilution" techniques that rely on 13 Cl abelling of selected amino acids.…”

“…[11] Because exchange between hyperpolarized HDO and amide H N protons only enhances al imited set of solvent-exposed residues under near-physiological conditions, [8] the 1 H- 15 Ncorrelation spectra are sparse and can be assigned despite the poor dispersion of their chemical shifts. [13] Felli and co-workers have advocated the use of 13 Cd irect detection techniques that allow the observation of IDPs under physiological conditions in 13 C-15 N correlation spectra but do not show any proton signals,which are the object of our work. [13] Felli and co-workers have advocated the use of 13 Cd irect detection techniques that allow the observation of IDPs under physiological conditions in 13 C-15 N correlation spectra but do not show any proton signals,which are the object of our work.…”

Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in E. coli‐Based Overexpression Systems

“…Additionally, the availability of isolated 13 C− 1 H spin pairs in histidine side chains allows for novel CPMG‐type experiments to probe enzyme fluctuations involving the motion of histidine side chains occurring on the milli‐ to microsecond timescales . Diverse isotopomers of our novel His‐precursor can be applied in overexpression media containing additional precursors for selective Phe, Tyr, and Trp labeling, which we recently developed –. We expect the corresponding spectra to feature well‐resolved NMR signals in the aromatic spectral region with optimized magnetization transfer pathways.…”