Modular protein engineering is a powerful approach for fabricating high-molecular-weight assemblies and biomaterials with nanoscale precision. Despite rapid advances in computational machine learning methods for designing proteins or topologically defined complexes, the construction of artificial structures that mimic natural architecture remains a significant challenge. Inspired by human muscle proteins, which exhibit a characteristic one-dimensional (1D) filamentous architecture and play a key role in maintaining the structural integrity and mechano-elasticity of muscle tissue, we focused on developing modular building blocks based on β-globular immunoglobulin and α-helical spectrin repeats from titin and dystrophin. The first strategy involves introducing circular permutation (CP) into β-globular titin domains. CP enables native interactions between the relocated donor β-strand and the exposed β-cleft, allowing the directional self-assembly of filamentous high-molecular-weight structures with a topology comparable to natural titin. To address robustness issues, we shifted our focus to dystrophin, composed of numerous tandem repeats of spectrin modules. Each module comprises a bundle of three α-helices, with the N- and C-termini located on opposite sides of the fold. Implementing precise protein design approaches, we engineered orthogonal building blocks from spectrin repeats and designed coiled-coil (CC) dimer-forming peptides. The CC peptides were precisely integrated to maintain the seamless continuity of the terminal spectrin α-helices, facilitating the formation of extended filaments. We demonstrated that this strategy leads to the effective construction of recombinant spectrin repeat-based filaments, which can assemble into bundled rigid rods, several micrometers in length (up to 15 µm). To our knowledge, this is the first strategy describing the engineering of high-molecular-weight rod-like structures from natural spectrin repeat-based building blocks derived from human dystrophin and designed CC coupling motifs. Furthermore, we showed that the coupling via CC motifs can be reversibly regulated by metal ions (Zn(II)) and chelators (EDTA). These rigid rods can also serve as a spatially organized scaffold for decorating proteins or biologically active peptides along their length with adjustable spacing ranging from ~35 to 75 ± 10 nm. These findings highlight the paramount potential of modular bottom-up protein engineering and tunable CCs for fabricating functionalized protein biomaterials for numerous biotechnological, biomedical, and bioengineering applications.
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