A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme
The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-13C]Glucose ([2-13C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-13C]pyruvate produced mainly by glycolysis and [3-13C]/[1,3-13C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs 13CÎ²-13CÎ³2 and 13CÎ±-13CÎ³1 in Val, and 13CÎ³-13CÎ´2 and 13CÎ²-13CÎ´1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin. âº Stereospecific assignments can improve the accuracy and precision of NMR structures. âº New strategy for solid-state NMR stereospecific resonance assignment of Val and Leu. âº Based on protein production in Escherichia coli media containing [2-13C]Glucose. âº Spectral simplification in the often congested methyl region. âº Applications to PrgI needles and ubiquitin are shown.