Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. However, little is understood about the importance of protein unfolding on the network assembly, severely limiting opportunities to utilize this nanoscale transition in the development of biomimetic and bioinspired materials. Here we control the force lability of a single protein building block, bovine serum albumin (BSA), and demonstrate that protein unfolding plays a critical role in defining the architecture and mechanics of a photochemically cross-linked native protein network. The internal nanoscale structure of BSA contains “molecular reinforcement” in the form of 17 covalent disulphide “nanostaples”, preventing force-induced unfolding. Upon addition of reducing agents, these nanostaples are broken rendering the protein force labile. Employing a combination of circular dichroism (CD) spectroscopy, small-angle scattering (SAS), rheology, and modeling, we show that stapled protein forms reasonably homogeneous networks of cross-linked fractal-like clusters connected by an intercluster region of folded protein. Conversely, in situ protein unfolding results in more heterogeneous networks of denser fractal-like clusters connected by an intercluster region populated by unfolded protein. In addition, gelation-induced protein unfolding and cross-linking in the intercluster region changes the hydrogel mechanics, as measured by a 3-fold enhancement of the storage modulus, an increase in both the loss ratio and energy dissipation, and markedly different relaxation behavior. By controlling the protein’s ability to unfold through nanoscale (un)stapling, we demonstrate the importance of in situ unfolding in defining both network architecture and mechanics, providing insight into fundamental hierarchical mechanics and a route to tune biomaterials for future applications.

Folded protein-based hydrogels are a novel class of biomaterials which combine the useful viscoelastic properties of individual proteins together with the prospect of rational design principles. Although the macroscopic properties of these materials have been well studied, there is a paucity of understanding of their mesoscopic formation mechanisms, especially given the differences in building blocks compared to biopolymer hydrogels. We present the results of a simulation study into the growth of polymeric networks of chemically cross-linked folded proteins that form the structural backbone of these hydrogels, observing how experimentally controllable parameters affect the resultant network growth and structural characteristics. We show that the initial volume fraction emerges as a dominant parameter at the network level but that the properties of the single protein remain important. We ultimately show that we can tune the properties of a monodisperse protein hydrogel network only within limits which are dictated primarily by implicit diffusion time scales.

Biological organisms make use of hierarchically organised structures to modulate mechanical behaviour across multiple lengthscales. We investigate this effect with a simulation study on globular domain polymers.

Fluctuating Finite Element Analysis (FFEA) is a software package designed to perform continuum mechanics simulations of proteins and other globular macromolecules. It combines conventional finite element methods with stochastic thermal noise, and is appropriate for simulations of large proteins and protein complexes at the mesoscale (length-scales in the range of 5 nm to 1 μm), where there is currently a paucity of modelling tools. It requires 3D volumetric information as input, which can be low resolution structural information such as cryo-electron tomography (cryo-ET) maps or much higher resolution atomistic co-ordinates from which volumetric information can be extracted. In this article we introduce our open source software package for performing FFEA simulations which we have released under a GPLv3 license. The software package includes a C ++ implementation of FFEA, together with tools to assist the user to set up the system from Electron Microscopy Data Bank (EMDB) or Protein Data Bank (PDB) data files. We also provide a PyMOL plugin to perform basic visualisation and additional Python tools for the analysis of FFEA simulation trajectories. This manuscript provides a basic background to the FFEA method, describing the implementation of the core mechanical model and how intermolecular interactions and the solvent environment are included within this framework. We provide prospective FFEA users with a practical overview of how to set up an FFEA simulation with reference to our publicly available online tutorials and manuals that accompany this first release of the package.

The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome.