Selective editing of Val and Leu methyl groups in high molecular weight protein NMR
The development of methyl-TROSY approaches and specific 13C–1H labeling of Ile, Leu and Val methyl groups in highly deuterated proteins has made it possible to study high molecular weight proteins, either alone or in complexes, using solution nuclear magnetic resonance (NMR) spectroscopy. Here we present 2-dimensional (2D) and 3-dimensional (3D) NMR experiments designed to achieve complete separation of the methyl resonances of Val and Leu, labeled using the same precursor, α-ketoisovalerate or acetolactate. The 2D experiment can further select the methyl resonances of Val or Leu based on the Cα or Cβ chemical shift values of Val or Leu, respectively. In the 3D spectrum, the methyl cross peaks of Val and Leu residues have opposite signs; thus, not only can the residue types be easily distinguished, but the methyl pairs from the same residue can also be identified. The feasibility of this approach, implemented in both 2D and 3D experiments, has been demonstrated on an 82 kDa protein, malate synthase G. The methods developed in this study will reduce resonance overlaps and also facilitate structure-guided resonance assignments.
KeywordsLeucine Valine Methyl Residue-type editing Malate synthase G
We thank Dr. Vitali Tugarinov (University of Maryland) for the MSG expression plasmid and Mr. Loren Colson for assistance with protein production. The research was supported by NIH grants R01GM074748 and R01GM086171, an AMMI grant, and the NMR core facility of City of Hope.
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