Chemical Protein Engineering: Backbone Cyclization Rescues Folding of a 183-Residue Truncated Domain of Malaria Parasite Protein PfAMA1.

Abstract

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.