Improved resolution and sensitivity of triple-resonance NMR methods for the structural analysis of proteins by use of a backbone-labeling strategy

A novel isotopic labeling strategy is described for the structural analysis of proteins by NMR. Overexpression of a protein in a mammalian cell-line cultured in a medium containing amino acids labeled only in the backbone (N, C-alpha, H-alpha, C') atoms leads to the formation of exclusively backbone-labeled protein. We demonstrate that the absence of the one bond scalar coupling between the C-13(alpha) and C-13(beta) atoms that is observed in uniformly C-13 enriched proteins offers a substantial sensitivity and resolution advantage in triple resonance NMR experiments that are commonly used to obtain backbone resonance assignments. This approach is illustrated in application to the beta subunit of human chorionic gonadotropin isotopically enriched with C-13 (97%), N-15 (97%), and H-2 (50%) exclusively in the backbone atoms of Phe, Val, and Leu residues.