Fig. 1. Identification of RINGPep1 from a SICLOPPS library. (a) Schematic of IDOL RTHS. The expression of the 434-IDOL fusion protein is induced by IPTG. IDOL dimerization leads to the formation of a functional repressor that prevents expression of the 3 reporter genes downstream: His3 (imidazole glycerol phosphate dehydratase), KanR (aminoglycoside 3′-phosphotransferase, kanamycin resistance), and LacZ (β-galactosidase). A cyclic peptide inhibitor of IDOL homodimerization will disrupt the repressor, leading to expression of the reporter genes and survival of the host colony on selective media. (b) Drop spotting analysis of RINGPep1 activity in the IDOL RTHS; serial dilutions (2.5 μL of ∼10ⁿ cells per mL) of the IDOL RTHS and the IDOL RTHS containing a SICLOPPS plasmid encoding RINGPep1. In the absence of IPTG and arabinose the strains have full growth capability, however upon addition of 25 μM IPTG (inducer of 434-IDOL) and 6.5 μM arabinose (inducer of SICLOPPS) the IDOL RTHS displays inhibition of growth, which is restored in the strain containing the plasmid encoding RINGPep1. (c) The residues on IDOL perturbed by RINGPep1 binding identified by ¹H ¹⁵N HSQC NMR are shown in blue (for spectrum see ESI Fig. 5,† for additional views of the structure, see ESI Fig. 6†). Each monomer of the IDOL homodimer is shown in a different shade of pink. (d) Assessing the binding of RINGPep1 to IDOL by MST reveals a K_d of 38.5 ± 3.0 μM. Data represented as mean ± SEM, n = 3.