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 c ultured 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 co upling between the C-13(alpha) and C-13(beta) atoms that is observed in uni formly C-13 enriched proteins offers a substantial sensitivity and resoluti on advantage in triple resonance NMR experiments that are commonly used to obtain backbone resonance assignments. This approach is illustrated in appl ication to the beta subunit of human chorionic gonadotropin isotopically en riched with C-13 (97%), N-15 (97%), and H-2 (50%) exclusively in the backbo ne atoms of Phe, Val, and Leu residues.