Improved Resolution and Sensitivity of Triple-Resonance NMR Methods for the Structural Analysis of Proteins by Use of a Backbone-Labeling Strategy

Abstract

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 α, H α, C‘) atoms leads to the formation of exclusively backbone-labeled protein. We demonstrate that the absence of the one bond scalar coupling between the 13 C α and 13 C β atoms that is observed in uniformly 13 C 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 β subunit of human chorionic gonadotropin isotopically enriched with 13 C (97%), 15 N (97%), and 2 H (50%) exclusively in the backbone atoms of Phe, Val, and Leu residues.