title: Selective editing of Val and Leu methyl groups in high molecular weight protein NMR.
abstract: The development of methyl-TROSY approaches and specific (13)C-(1)H 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 C(α) or C(β) 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.
authors.affiliation: Department of Immunology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.
authors.forename: Weidong
authors.initials: W
authors.lastname: Hu
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Namanja
authors.forename: Steven
authors.initials: S
authors.lastname: Wong
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
mesh_terms: D002247:Carbon Isotopes; D029968:Escherichia coli Proteins / Q000737:chemistry; D007930:Leucine / Q000737:chemistry*; D008292:Malate Synthase / Q000737:chemistry; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D014633:Valine / Q000737:chemistry*
chemical_list: D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D011506:Proteins; D008292:Malate Synthase; D007930:Leucine; D014633:Valine