Conformation and Dynamics of Monomeric, Phase-Separated, and Cross-Linked Resilin Biomaterials

Resilin is an elastomeric protein found in insects that provides flexibility and locomotive function in numerous biological contexts. Recombinant resilin and resilin-derived proteins share resilin’s capacity for liquid–liquid phase separation (LLPS) and formation of materials with high elasticity and biocompatibility, making it a promising candidate for regenerative medicine and tissue engineering applications. While prior research has focused on domain 1 of resilin, the role of domain 3 (D3) in resilin self-assembly and material properties is not well understood. Here, we used nuclear magnetic resonance, electron paramagnetic resonance, and small-angle X-ray scattering to study the conformation, dynamics, and intermolecular interactions of D3 as a monomer, in the phase-separated state, and as a cross-linked gel. We show that D3 remains unusually dynamic and is primarily disordered in all three states. In elucidating the mechanism of D3 LLPS, we find a complex set of electrostatic and π-based interactions complemented by the hydrophobic effect that finely tune the solution sensitivity of D3 and its capacity for LLPS. Overall, these results highlight the complex mechanisms governing resilin LLPS with implications for utilizing resilin-derived sequence features in the rational design of self-assembling biomaterials.