A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated ¹⁵N-, ¹³C-, ²H-labeled proteins

A selective protonation strategy is described that uses [3-²H] ¹³C α-ketoisovalerate to introduce (¹H-δ methyl)-leucine and (¹H-γ methyl)- valine into ¹⁵N-, ¹³C-, ²H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of ≃100 mg/L α-ketoisovalerate in the bacterial growth medium. Addition of [3,3-²H₂] α-ketobutyrate to the expression media (D₂O solvent) results in the production of proteins with (¹H-δ1 methyl)- isoleucine (>90% incorporation), ¹H-¹³C HSQC correlation spectroscopy establishes that CH₂D and CHD₂ isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.