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

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 13C–1H spin systems surrounded by 12C and 2H. 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. Selective Trp‐labeling: Tryptophans are among the most conserved residues at protein “hot‐spots”. In order to study their prominent role in protein binding events by NMR spectroscopy, indole Trp‐precursors were synthetized with isolated 13C–1H spin systems, ideally suited for the analysis of side‐chain dynamics. A novel α‐ketoacid Trp‐precursor for selective backbone isotope labeling is also presented.