Coupling of Spectrin\nRepeat Modules for the Assembly\nof Nanorods and Presentation\nof Protein Domains

Modular protein engineering is a powerful approach for\nfabricating\nhigh-molecular-weight assemblies and biomaterials with nanoscale precision.\nHerein, we address the challenge of designing an extended nanoscale\nfilamentous architecture inspired by the central rod domain of human\ndystrophin, which protects sarcolemma during muscle contraction and\nconsists of spectrin repeats composed of three-helical bundles. A\nmodule of three tandem spectrin repeats was used as a rigid building\nblock self-assembling via coiled-coil (CC) dimer-forming peptides.\nCC peptides were precisely integrated to maintain the spectrin α-helix\ncontinuity in an appropriate frame to form extended nanorods. An orthogonal\nset of customizable CC heterodimers was harnessed for modular rigid\ndomain association, which could be additionally regulated by metal\nions and chelators. We achieved a robust assembly of rigid rods several\nmicrometers in length, determined by atomic force microscopy and negative\nstain transmission electron microscopy. Furthermore, these rigid rods\ncan serve as a scaffold for the decoration of diverse proteins or\nbiologically active peptides along their length with adjustable spacing\nup to tens of nanometers, as confirmed by the DNA-PAINT super-resolution\nmicroscopy. This demonstrates the potential of modular bottom-up protein\nengineering and tunable CCs for the fabrication of functionalized\nprotein biomaterials.