An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated ¹⁵N,¹³C-laheled proteins

A biosynthetic strategy has recently been developed for the production of ¹⁵N,¹³C,²H-labeled proteins using ¹H₃C-pyruvate as the sole carbon source and D₂O as the solvent. The methyl groups of Ala, Val, Leu and Ile (γ2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a ¹⁵N,¹³C,²H,¹H₃C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30-40 kDa molecular weight range.