Fig. 5. Structural and functional characterization studies of CPPC–DRPs binding CD28. (a) Binding of reduced and oxidized drp8 labeled with fluorescein (FITC) to CD28 determined using a fluorescence polarization (FP) assay. (b) Binding of oxidized drp1, drp7, drp8 and drp9 to CD28 determined by competition with oxidized FITC-drp8 in a FP assay. Note that drp1, drp7, drp8, drp9, and FITC-drp8 used for FP assay and structural characterization studies have been purified to a purity of >95% using HPLC (Fig. S20 and S21†). (c) Solution structures of drp8; the lowest-energy NMR structure of drp8-I and drp8-II and the respective ensemble of the 15 lowest-energy structures. (d) Confocal fluorescence images of FITC-drp8 bound on the cell surfaces. Top: HEK293T cells with recombinant mCherry-CD28 (CD28(+); top) and mCherry (CD28(−); bottom) expressed on the cell surfaces were incubated with 1 μM FITC-drp8 in a medium for 30 min. Green fluorescence was from FITC-drp8; yellow fluorescence was from the mCherry protein.