¹⁵N-labelled proteins by cell-free protein synthesis: Strategies for high-throughput NMR studies of proteins and protein-ligand complexes

[¹⁵N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [¹⁵N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [¹⁵N]-HSQC spectra by amino acid type selective [¹⁵N]-labelling. The samples can be measured without chromatographic protein purification, dilution of isotopes by transaminase activities are suppressed, and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum number of samples for the identification of all [¹⁵N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [¹⁵N]-HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular, combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h, including protein production and NMR measurement.