Coiled Coil Peptide\nTiles (CCPTs): Expanding the Peptide\nBuilding Block Design with Multivalent Peptide Macrocycles

Owing to their synthetic accessibility and protein-mimetic\nfeatures,\npeptides represent an attractive biomolecular building block for the\nfabrication of artificial biomimetic materials with emergent properties\nand functions. Here, we expand the peptide building block design space\nthrough unveiling the design, synthesis, and characterization of novel,\nmultivalent peptide macrocycles (96mers), termed coiled coil peptide\ntiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide\ndomains that are separated by flexible linkers. The constraints, imposed\nby cyclization, confer CCPTs with the ability to direct programmable,\nmultidirectional interactions between coiled coil-forming “edge”\ndomains of CCPTs and their free peptide binding partners. These fully\nsynthetic constructs are assembled using a convergent synthetic strategy\nvia a combination of native chemical ligation and Sortase A-mediated\ncyclization. Circular dichroism (CD) studies reveal the increased\nhelical stability associated with cyclization and subsequent coiled\ncoil formation along the CCPT edges. Size-exclusion chromatography\n(SEC), analytical high-performance liquid chromatography (HPLC), and\nfluorescence quenching assays provide a comprehensive biophysical\ncharacterization of various assembled CCPT complexes and confirm the\northogonal colocalization between coiled coil domains within CCPTs\nand their designed on-target free peptide partners. Lastly, we employ\nmolecular dynamics (MD) simulations, which provide molecular-level\ninsights into experimental results, as a supporting method for understanding\nthe structural dynamics of CCPTs and their complexes. MD analysis\nof the simulated CCPT architectures reveals the rigidification and\nexpansion of CCPTs upon complexation, i.e., coiled coil formation\nwith their designed binding partners, and provides insights for guiding\nthe designs of future generations of CCPTs. The addition of CCPTs\ninto the repertoire of coiled coil-based building blocks has the potential\nfor expanding the coiled coil assembly landscape by unlocking new\ntopologies having designable intermolecular interfaces.