Capturing the impact of protein unfolding on the dynamic assembly of protein networks.

Abstract

The rapid assembly of molecular or nanoscale building blocks into extended arrays is crucial to the construction of functional networks <i>in vivo</i> and <i>in vitro</i> and depends on various factors. One factor seldom considered is the dynamic changes of the building block shape. Folded protein building blocks offer a unique system to investigate dynamic shape changes due to their intrinsic ability to change from a compact and specific folded structure to an extended unfolded structure in response to a perturbation such as force. Here, we use photochemically crosslinked folded protein hydrogels constructed from force labile protein building blocks as a model dynamic shape-changing network system and characterise them by combining time-resolved rheology and small-angle X-ray scattering (SAXS). This approach probes both the load-bearing network structures, using rheology, and network architectures, using SAXS, thereby providing a crosslength scale understanding of the network formation. We propose a triple assembly model for the structural evolution of networks constructed from force labile protein building block consisting of: primary formation where monomeric folded proteins create the preliminary protein network scaffold; a subsequent secondary formation phase, where larger oligomers of protein diffuse to join the preliminary network scaffold; and finally <i>in situ</i> unfolding and relaxation which leads to the mature network structure of connected larger and denser fractal-like clusters. The time-resolved SAXS data provides evidence that protein unfolding occurs on the edges of the fractal-like clusters, resulting in a population of unfolded proteins in the space between clusters. Identifying the key stages of assembly in protein networks constructed from force labile proteins provides a greater understanding of the importance of protein unfolding in hierarchical biomechanics <i>in vivo</i> and creates future opportunities to develop bespoke biomaterials for novel biomedical applications.