Figure 3.

Inhibition of D₃D₄ ankyrin binding to cdB3 by increasing concentrations of the peptide comprising residues 63–73 (loop 1) of cdB3. A loop 1 mimetic peptide was synthesized from residues 63–73 of cdB3 by modifiying the peptide to contain a cysteine residue at each end. A cyclic form of the peptide was formed by air oxidation followed by C₁₈ reverse phase HPLC, while the linearized form was obtained by incubating the cyclized peptide in 1 mM dithiothreitol. Both peptide compositions were confirmed by mass spectrometry. After incubation of D₃D₄ ankyrin and cdB3 (His-tagged) in the presence of increasing concentrations of peptide, complexes were captured on Co²⁺-NTA beads, washed and eluted as before, and assayed for bound GST-D₃D₄ ankyrin by analysis of GST activity. Error bars correspond to S.D., where n=2.