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

1999; American Chemical Society; Volume: 121; Issue: 50 Linguagem: Inglês

10.1021/ja993083w

1943-2984

Philip E. Coughlin, Frank E. Anderson, Ed J. Oliver, Jonathan M. Brown, Steve W. Homans, Susan Pollak, Joyce W. Lustbader,

Protein Structure and Dynamics

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, Cα, Hα, C') atoms leads to the formation of exclusively backbone-labeled protein. We demonstrate that the absence of the one bond scalar coupling between the 13Cα and 13Cβ atoms that is observed in uniformly 13C 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 13C (97%), 15N (97%), and 2H (50%) exclusively in the backbone atoms of Phe, Val, and Leu residues.