Figure 4.

(a) Schematic diagram of amino acid biosynthesis and methyl group-specific ¹³C-labeling. Ketobutyrate and ketoisovalerate, precursors of isoleucine and valine/leucine, respectively, are utilized for methyl group-selective ¹³C-labeling of isoleucine, valine, and leucine residues. The red colored carbons are from same origin in metabolism. The blue asterisks denoted on the isoleucine indicate the carbons from pyruvate; (b) Chemical shift perturbation method as a protein-based NMR approaches for SBDD studies. Black spheres on the protein, represented with ribbon diagram, indicate position of ¹³C-labeled methyl groups. Ligand is represented with stick diagram. Numbers on the spheres correspond to each ¹H-¹³C correlation NMR signal on the right panel. Chemical shift perturbation induced by interaction with ligand is indicated by gray arrows.