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.