Figure 1.

Orthogonal CC peptide pairs with K_d in the nanomolar range based on modifications at the b, c, and f sites. (A, C) Thermal denaturation profiles of peptides (40 µM; magenta and cyan) and CCs (20 µM each peptide; black) monitored by a CD signal at 222 nm. The midpoint Tm was calculated based on thermodynamic model fit³⁵. (B, D) Isothermal titration calorimetry (ITC) analysis of the binding affinity of designated CC peptide pairs. The binding isotherms of heat release per injection are depicted as a function of the increasing peptide-to-peptide molar ratio. The dissociation constant, K_d^ITC, was calculated using the two-state dimer association model. (E) Heat map of the matrix of the calculated midpoint Tm from thermal denaturation scans of all peptide combinations. (F) Scheme of reconstitution of CC-split luciferase managed by a CC-forming peptide pair. (G) Luciferase activity of reconstituted CC-split luciferase in HEK293T measured 48 h after transfection of HEK293T cells with a plasmid expressing a combination of nLuc tethered to N8 (30 ng) and cLuc tethered to N7 peptide (30 ng). (H, I) Luciferase activity determined 48 h after transformation of HEK293T cells with plasmids expressing nLuc:N8 or nluc:N6 (30 ng); and cLuc tethered to N7 or P7A or cLuc tethered to N5 and P5A (10–70 ng). (J) Orthogonality of designed N peptide set in HEK293T cells by co-transfection of the nLuc:N8 or nLuc:N6 fusion encoding plasmid (30 ng), and cLuc:CC (CC stands for N5, P5A, N7, P7A) (30 ng). Reconstituted luciferase activity was measured 48 h after transfection. Amounts of plasmids are indicated in Table S2. The values (G-J) represent the means (± s.d.) from four independent cell cultures, individually transfected with the same mixture of plasmids, and are representative of two independent experiments.