Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Abstract
Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L and V are stereo-specifically labeled. We show that 2-ketobutyrate cannot be combined with Ala and 2-acetolactate (for the stereo-specific labeling of L and V) as this results in co-incorporation incompatibility and isotopic scrambling. Thus, we developed a robust and cost-effective enzymatic synthesis of the isoleucine precursor, 2-hydroxy-2-(1â?²-[2H2], 2â?²-[13C])ethyl-3-keto-4-[2H3]butanoic acid, as well as an incorporation protocol that eliminates metabolic leakage. We show that application of this labeling scheme to a large 82Â*kDa protein permits the detection of long-range 1Hâ??1H NOE cross-peaks between methyl probes separated by up to 10Â*Ã?.
Source: Journal of Biomolecular NMR