Chemical Protein Engineering: Backbone Cyclization Rescues Folding of a 183‐Residue Truncated Domain of Malaria Parasite Protein <i>Pf</i>AMA1

The interaction between apical membrane antigen 1 (PfAMA1) and rhoptry neck protein 2 (PfRON2) is crucial for Plasmodium falciparum red blood cell invasion, making it a key target for anti-malarial drug development strategies. Here, we report the chemical synthesis of PfAMA1 domain I (PfAMA1-DI) in both linear and backbone-circularized forms, employing a six-segment convergent synthesis approach exploiting one-pot chemistries and solubilizing tags. The chemically synthesized linear PfAMA1-DI construct exhibited incomplete disulfide bond formation during folding, likely due to increased terminal flexibility in the absence of domain II. To address this, we employed backbone cyclization of the large 180-residue polypeptide chain, with 3-residue linker sequence, as a unique strategy to conformationally restrict its termini and facilitate correct disulfide bond formation. Introducing a multipurpose affinity and solubility tag to the cyclicPfAMA1-DI construct further improved the folding yield by mitigating aggregation. The predicted structure using ColabFold-Alphafold2 indicated that PfRON2 ligand binds within the hydrophobic groove of the cyclicPfAMA1-DI construct similar to the native interactions. These findings underscore the potential of large protein backbone cyclization to stabilize protein structure, offering a compelling strategy for the chemical synthesis of otherwise unstable protein domains with broad applications in miniature protein engineering.