Fig. 3. Structure-based applications of CPPC–DRPs. (a) Flow diagram illustrating the applications that can be explored on the basis of structures of CPPC–DRPs, including design of CPPC–DRPs with new structures and functions through epitope grafting and selection of new CPPC–DRPs through library design and screening. (b) Illustration of the design of drp4. From left to right: drp1 as a scaffold shown in cyan; the structural motif of PPα-Tyr (orange) grafted into the drp1 scaffold; structures of the finally designed drp4 (ensemble of the 15 lowest-energy structures; PDB 7W8Z). (c) Schematic view of epitope grafting and design of a secondary library to generate new CPPC–DRPs with Keap1-binding capability. drp5 is generated by grafting an epitope (sequence: DEETGE) into the drp1 scaffold. A secondary library was designed (detailed in Table S1†) by randomizing the residues flanking the grafted epitope. drp6 was the most abundantly enriched sequence from the library screening against Keap1 (dataset S3 and Fig. S9†). (d) SPR sensorgrams showing interaction of Keap1 with the two oxidized products of drp5 (drp5-a and drp5-b) in a concentration-dependent manner (top), and the equilibrium dissociation constant (KD) values of drp5-a and drp5-b toward Keap1 calculated from SPR measurements (bottom). (e) HPLC chromatogram showing the oxidation of drp6 (50 μM) in a phosphate buffer (pH 7.4, 100 mM) containing 0.5 mM GSSG and 6 M Gu·HCl; the lowest-energy NMR structure of drp6 and the ensemble of the 15 lowest-energy structures; SPR sensorgrams determining the KD of drp6 toward Keap1. Note that drp5-a, drp5-b and drp6 used for SPR measurements and structural characterization studies have been purified to a purity of >95% using HPLC (Fig. S10†). Different N-terminal residues were incorporated into the peptides to facilitate synthesis or analysis.
