Selective backbone labelling of ILV methyl labelled proteins

Adding the C-13 labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of C-12 labelled glucose instead of the commonly used (D-2, C-13) glucose leads not only to the C-13 labelling of (I, L, V) methyls but also to the selective C-13 labelling of the backbone C-alpha and CO carbons of the Ile and Val residues. As a result, the backbone (H-1, N-15) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue NOE effect, and should therefore be applicable to larger systems.