Engineered Recombinant\nHagfish Intermediate Filament\nProteins: Unraveling Domain Roles in Synthetic Fiber Formation and\nMechanics

Hagfish intermediate filament (HIF) proteins, consisting\nof α\nand γ subunits, have been previously recombinantly expressed,\npurified, and utilized to form dry fibers with impressive mechanical\nproperties. HIFα and HIFγ consist of three protein domains\n(N-termini, C-termini, and central rod domain). To begin to understand\nthe structure–function relationship between the protein domains\nin fiber formation and properties in a synthetic fiber spinning system,\nwe designed recombinant protein constructs with varying combinations\nof the N-terminus, central rod domain (CRD), and C-terminus for both\nthe α and γ proteins. The constructs, for both α\nand γ, were expressed, purified, and spun into dry fibers, which\nwere then tested and analyzed for mechanical and structural properties.\nMechanical testing revealed that the α constructs had the highest\ntensile strength when both termini were removed while including either\nterminus improved strain and toughness compared to α CRD constructs.\nThe γ constructs displayed improved tensile strength and elastic\nmodulus when only the N-terminus was present. Mixing the α and\nγ constructs generally enhanced the mechanical properties compared\nto the full-length rHIFα and rHIFγ. Fourier transform\ninfrared-attenuated total reflection (FTIR-ATR) analysis indicated\nthat the CRD contributes more to the β-sheet content in the\nstretched fibers, while the termini contribute more to the α-helical/random\ncoil regions. These findings provide valuable insights into the roles\nof the different protein domains in the assembly and mechanical performance\nof rHIF and other recombinantly expressed IF. By understanding these\nstructure–function relationships, functionally tailored recombinant\nIF proteins can be designed for specific applications in biomaterials\ndevelopments.