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

5 years ago

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

Roman J. Lichtenecker, Gerald Platzer, Leonhard Geist, Julia Schörghuber, Robert Konrat

The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever-expanding owing 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 methodological gap, we present a novel metabolic precursor for cell-based protein overexpression to assemble 13C/2H isotope patterns in the peptide backbone, as well as in side chain positions of a mechanistically distinguished histidine residue. For histidines only! The enol tautomer of imidazole-4-pyruvic acid is introduced as a novel precursor in E. coli-based labeling of histidine residues. A straightforward synthetic route allows for selective 13C-labeling at the ϵ1 imidazole side-chain position, thus providing a valuable tool to study histidine's dynamic properties, pKa values, and tautomer populations by protein NMR spectroscopy.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/cbic.201700192