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.
- Multidimensional NMR
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