Design of proteins using rigid organic macrocycles as scaffolds Original Research Article

We have designed and synthesized new three-helix template-assembled synthetic proteins (TASPs) 1a–c. The template was the rigid cyclotribenzylene (CTB) macrocycle 2, which has C3 symmetry. Thiol moieties on the CTB template were used to link cysteine-containing peptide strands 3a–c via disulfide bonds. With designed peptide strands of 15 and 18 residues in length, the structure of TASPs 1a–c were determined to be helical in water according to circular dichroism (CD) spectroscopy. The helicities of TASPs 1a–c were unchanged over large ranges of pH (2–12) and salt concentrations (0–2 M KCl). TASPs 1a–c were also extremely resistant to chemical denaturants: it requires a guanidine hydrochloride (GnHCl) concentration of 7.4 M for TASPs 1a–c to lose 50% of their helicity. The major force for stabilization of TASPs 1a–c is the hydrophobic bundling of the helices.