Selective 'unlabeling' of amino acids in fractionally 13C labeled proteins:: An approach for stereospecific NMR assignments of CH3 groups in Val and Leu residues

A novel methodology for stereospecific NMR assignments of methyl (CH3) groups of Val and Leu residues in fractionally C-13-labeled proteins is presented. The approach is based on selective 'unlabeling' of specific amino acids in proteins while fractionally C-13-labeling the rest. A 2D [C-13-H-1] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the 'unlabeled' amino acid residues. Such spectral simplification aids in unambiguous stereospecific assignment of diastereotopic CH3 groups in Val and Leu residues in large proteins. This methodology has been demonstrated on a 15 kDa calcium binding protein from Entamoeba histolytica (Eh-CaBP).