Protein nanostructures with precisely defined geometries have many potential applications in catalysis, sensing, signal processing, and drug delivery. While many de novo protein nanostructures have been assembled via non-covalent intramolecular and intermolecular interactions, a largely unexplored strategy is to construct nanostructures by covalently linking multiple individually folded proteins through site-specific ligations. Here, we report the synthesis of single-chain protein nanostructures with triangular and square shapes made using multiple copies of a three-helix bundle protein and split intein chemistry. Coarse-grained simulations confirm the experimentally observed flexibility of these nanostructures, which is optimized to produce triangular structures with high regularity. These single-chain nanostructures also display ultra-high thermostability, resist denaturation by chaotropes and organic solvents, and have applicability as scaffolds for assembling materials with nanometer resolution. Our results show that site-specific covalent ligation can be used to assemble individually folded proteins into single-chain nanostructures with bespoke architectures and high stabilities.
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