Proline is unique among the canonical amino acids: its pyrrolidine side chain biases the conformational space accessible to this residue. Non-canonical proline analogs can modulate the cis-trans isomerization and ring puckering properties of proline, and can contribute additional chemical functionality while maintaining the pyrrolidine side chain. Thus, non-canonical proline residues can be used to interrogate the role of proline’s conformation in protein structure and function, and are useful tools in protein engineering endeavors. Recombinant insulin is a life-saving therapeutic in the treatment of diabetes mellitus. A number of FDA-approved insulin variants that enable improved control of blood glucose have been achieved through standard mutagenesis approaches. For instance, the fast-acting insulin lispro contains two point mutations (ProB28Lys and LysB29Pro) that destabilize insulin oligomers, leading to faster absorption. However, even fast-acting insulins are delayed when compared to the insulin-action profile of a healthy pancreas, and insulins undergo irreversible denaturation upon storage. Here, we widen the scope of proline analogs that are amenable to residue-specific incorporation into recombinant proteins in E. coli. We replace ProB28 of human insulin, and ProB29 of insulin lispro, with non-canonical proline residues, and find that the resulting insulin variants exhibit altered oligomerization and fibrillation lag times in vitro. These results demonstrate how subtle molecular changes through non-canonical amino acid mutagenesis can be used to engineer therapeutically-relevant properties of protein drugs.

Incorporation of non-canonical proline residues into proteins in Escherichia coli

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