Engineered Recombinant Hagfish Intermediate Filament Proteins: Unraveling Domain Roles in Synthetic Fiber Formation and Mechanics.

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