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

The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever-expanding due to progress in experimental techniques, hardware development and novel labeling approaches. Multiple sophisticated methods of aliphatic residue labeling can be found in the literature, whereas the selective incorporation of NMR active isotopes into other amino acids still holds the potential for improvement. In order to close this methodical gap, we present a novel metabolic precursor for cell-based protein overexpression to assemble 13C/2H isotope patterns in backbone, as well as in side chain positions of the mechanistically distinguished histidine residue. We tested our new method in the overexpression of three model proteins. The resulting NMR spectra displayed no interference to other metabolic pathways of the overexpressing E.coli strain and very high isotope incorporation rates. We additionally elaborated a straightforward synthetic route to access an isotopologue, which allows for the introduction of 13C into the imidazole side-chain. This labeling pattern represents a valuable tool for diverse NMR experiments to probe for histidine side-chain dynamics, tautomeric distributions and pKa-values. In a first application, we labeled a domain of the oncogenic transcription factor v-Myc and investigated chemical shift perturbations of the corresponding histidine signals upon addition of the protein interaction partner Max.