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. Time‐resolved structural measurements reveal the presence of two distinct formation modes in folded protein networks: Primary Formation (purple) of the preliminary network resulting from the diffusion of protein building blocks; and Secondary Formation (dark yellow) densifying the network via the slower diffusion of high‐order cross‐linked protein oligomers formed during the primary formation, joining the network and the formation of ‘intra’‐network cross‐links.