Fig. 4. Application of the valine-selective decoupling pulse on biologically relevant proteins.

Constant-time 2D ¹H–¹³C methyl HMQC spectra of four biologically relevant proteins are shown. a Cas9 HNH domain (130 µM, 15 kDa); b Cas9 REC1-2 domain (250 µM, 52 kDa); c Human IL2 (920 µM, 15.4 kDa); d eIF4A (50 µM, 46 kDa). Unambiguous distinction between the valine (blue) and leucine (red) resonances is achieved. Only Leu–Val overlapping region of the HMQC spectrum is shown here. The full spectra of these proteins are presented in Supplementary Figs. 6 and 7. e Here we show the 3D structure for the REC1-2 domain of Cas9 (PDB ID: 4UN3)⁵² where the valine and leucine residues are highlighted in blue and red, respectively.