Coiled Coil Peptide Tiles (CCPTs): Multivalent Peptide Macrocycles for Expanding the Coiled Coil Assembly Toolkit

As one of the most well-understood protein folds, coiled coils represent an attractive assembly directing motif for engineering modular and responsive bionanomaterials. Here, we expand the coiled coil assembly “toolkit” through unveiling the design and synthesis of novel, multivalent peptide macrocycles (96mers) that comprise multiple orthogonal coiled coil peptide domains. These fully synthetic constructs, termed coiled coil peptide tiles (CCPTs), are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along CCPT edges. Size exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide comprehensive biophysical characterization of various CCPT complexes and confirm the orthogonal co-localization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamic (MD) simulations, which provide molecular-level insights to experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveal the atomic-level interactions mediating their structure and stability and provide insights for guiding designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks have the potential for expanding the coiled coil assembly landscape by unlocking new topologies through designable intermolecular interfaces.