An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein

A strategy for the introduction of (H-1,C-13-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D2O based bacterial growth medium supplemented with 800 mg/l of 2-[H-2], 3-[C-13] l-alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of alpha-ketoisovalerate-d(7), succinate-d(4) and l-isoleucine-d(10) with labeled l-alanine, reduces undesired background labeling to < 1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods.