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

A biosynthetic strategy has recently been developed for the production of N-15,C-13,H-2-labeled proteins using (H3C)-H-1-pyruvate as the sole carbon source and D2O as the solvent. The methyl groups of Ala, Val, Leu and Ile (gamma 2 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 N-15,C-13,H-2,(H3C)-H-1-labeled SH2 domain at 3 degrees C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30-40 kDa molecular weight range.