Capturing Dynamic Assembly of Nanoscale Proteins During Network Formation.

The structural evolution of hierarchical structures of nanoscale biomolecules is crucial for the construction of functional networks in vivo and in vitro. Despite the ubiquity of these networks, the physical mechanisms behind their formation and self-assembly remains poorly understood. Here, this study uses photochemically cross-linked folded protein hydrogels as a model biopolymer network system, with a combined time-resolved rheology and small-angle x-ray scattering (SAXS) approach to probe both the load-bearing structures and network architectures respectively thereby providing a cross-length scale understanding of the network formation. Combining SAXS, rheology, and kinetic modeling, a dual formation mechanism consisting of a primary formation phase is proposed, where monomeric folded proteins create the preliminary protein network scaffold; and a subsequent secondary formation phase, where both additional intra-network cross-links form and larger oligomers diffuse to join the preliminary network, leading to a denser more mechanically robust structure. Identifying this as the origin of the structural and mechanical properties of protein networks creates future opportunities to understand hierarchical biomechanics in vivo and develop functional, designed-for-purpose, biomaterials.