Figure 2. MAS-DNP methods for probing protein-ligand binding.

(A) Yeast-based ¹³C labeling of cholesterol using site-specifically ¹³C-labeled glucose. The labeled carbon sites on cholesterol are in red and blue for cholesterols produced from 1-¹³C and 2-¹³C glucose molecules, respectively. (B) A structural model of a cholesterol molecule bound to the influenza M2 proteins. The key Ile and Phe residues, as well as their distances to cholesterol carbons, are shown. (C) Signal bleaching quantified in solution ¹H-¹⁵N HSQC spectra due to the binding of radicals to dihydrofolate reductase. (D) A model of E. coli dihydrofolate reductase with DNP bleaching information represented by the intensity ratios of ¹³C-¹³C DARR spectra collected on two samples containing either bound radicals or exogenous radicals. (E) Scheme for incorporating a carbohydrate ligand to a paramagnetic tag for selective DNP. (F) Selective DNP ¹³C–¹³C INADEQUATE difference spectrum of LecA obtained using k=1: only the tightly bound residues are observed. (G) Sideview of LecA. Residues observed using selective DNP are highlighted, with the corresponding k values given. Figures 2A-2D are adapted from references [38, 51] with copyright permission. Figures 2E-2G are adapted from reference [31], an open-access article.