Selective backbone labelling of ILV methyl labelled proteins

Abstract

Adding the 13C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of 12C labelled glucose instead of the commonly used (2D, 13C) glucose leads not only to the 13C labelling of (I, L, V) methyls but also to the selective 13C labelling of the backbone C(alpha) and CO carbons of the Ile and Val residues. As a result, the backbone (1H, 15N) 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.