*4.3. Peptide–RTA Interaction*

The peptide–RTA interaction was measured by surface plasmon resonance (SPR) using Biacore T200. The untagged RTA was immobilized on a CM5 chip at 2000 to 3000 RU by amine coupling. The reference channel was blocked as the active channel. The peptides were passed over the surface at 6.2, 18.5, 55.6, 166.7, and 500 μM at a flow rate of 30 μL/min. The running buffer was 10 mM Tris-HCl pH7.5, 60 mM KCl, 10 mM MgOAc, and 0.5% DMSO. The affinity was obtained by fitting the binding data using Biacore T200 Evaluation Software version 3.0.

The peptide–RTA mutant interactions were measured using Biacore T200. The 10×His-tagged wild-type RTA or the 10×His-tagged RTA mutants were captured on an NTA chip to around 2000 RU. The blank surface without Ni+ was used as the reference. The peptides were passed over the surface at 30 μL/min. The running buffer was the same as the untagged RTA. The binding signals were normalized for the differences in surface density.
