Methyl and Amino Acid-Type Labeling
Protonated carbon-13 methyl groups in deuterated proteins are excellent probes for structure and dynamics, and are crucial when studying high-molecular-weight protein and protein complexes. Methyl labeling refers to methods that introduce either 13CH3 or 13CHD2 groups into otherwise a uniform deuterated protein. Methyl labeling can be achieved in both prokaryotic and eukaryotic expression systems by adding the amino acids(s) in sufficient amounts directly to the cell culture medium prior to protein induction. For E. coli cultures, selective protonation on methyl groups of isoleucine, leucine, and valine (ILV) can be achieved by adding α-keto acid precursors to D2O-based cell culture medium one hour prior to induction. α-Ketoisobutyrate becomes converted to isoleucine, and α-ketoisovaleric acid becomes converted to leucine and valine. This methodology has dramatically increased the useful molecular weight range of protein and protein complexes assessable by NMR when using methyl TROSY techniques.