Cross-Linking and Bundling of Self-Assembled Protein-Based Polymer Fibrils via Heterodimeric Coiled Coils

Previously, we developed triblock protein polymers that form fibrillar hydrogels at low protein polymer concentrations (denoted C₂-S^H₄₈-C₂). We here demonstrate that the structure of these hydrogels can be tuned via heterodimeric coiled coils that cross-link and bundle the self-assembled protein polymer fibrils. We fused well-characterized, 47 amino acids-long heterodimeric coiled coil "linkers" (D^A or D^B) to the C-terminus of the triblock polymer. The resulting C₂-S^H₄₈-C₂-D^A and C₂-S^H₄₈-C₂-D^B polymers, were successfully produced as secreted proteins in Pichia pastoris, with titers of purified protein in the order of g L^-1 of clarified broth. Atomic force microscopy showed that fibrils formed by either C₂-S^H₄₈-C₂-D^A or C₂-S^H₄₈-C₂-D^B alone already displayed extensive bundling, apparently as a result of homotypic (D^A/D^A and D^B/D^B) interactions. For fibrils prepared from protein polymers having no linkers, plus a small fraction of polymers containing either D^A or D^B linkers, no cross-linking and bundling was observed. At these same low concentrations of linkers, fibrils containing both the D^A and the D^B linkers did show cross-linking and bundling as a consequence of heterodimer formation. This work shows that we can control the extent of bundling and cross-linking of supramolecular fibrils by varying the density of heterodimerizing coiled coils in the fibrils, which is promising for the further development of materials that mimic the extracellular matrix.