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-(13)C]Glucose ([2-(13)C]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-(13)C]pyruvate produced mainly by glycolysis and [3-(13)C]/[1,3-(13)C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs (13)Cβ-(13)Cγ2 and (13)Cα-(13)Cγ1 in Val, and (13)Cγ-(13)Cδ2 and (13)Cβ-(13)Cδ1 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.
Max Planck Institute for Biophysical Chemistry, Department of NMR-based Structural Biology, Am Fassberg 11, 37077 Göttingen, Germany.
Guohua
G
Lv
Hannes Klaus
HK
Faßhuber
Antoine
A
Loquet
Jean-Philippe
JP
Demers
Vinesh
V
Vijayan
Karin
K
Giller
Stefan
S
Becker
Adam
A
Lange
D002247:Carbon Isotopes / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006019:Glycolysis; D007930:Leucine / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010427:Pentose Phosphate Pathway; D011487:Protein Conformation; D019289:Pyruvic Acid / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*; D014633:Valine / Q000737:chemistry*
D002247:Carbon Isotopes; D025801:Ubiquitin; D019289:Pyruvic Acid; D007930:Leucine; D014633:Valine; D005947:Glucose