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 sol e carbon source and D2O as the solvent. The methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated, while the remain ing positions in the molecule are largely deuterated. An (H)C(CO)NH-TO CSY experiment is presented for the sequential assignment of the proto nated 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 tim e 18.8 ns), demonstrating the utility of the method for proteins in th e 30-40 kDa molecular weight range.