Abstract
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.
Highlights
Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind
We present a combined experimental and modeling approach to show that in situ unfolding of bovine serum albumin (BSA) modulates the BSA hydrogel network architecture, in particular the intercluster region which demonstrably dominates the mechanical response of the hydrogel network
The covalent staples are mechanically robust and capable of withstanding forces up to 2nN,[43,44] which is greatly in excess of the 20−100 pN thought to be generated in the cross-linked protein network,[12,45] yet these bonds are rapidly removed by reducing agents such as dithiothreitol (DTT) used in this study
Summary
Hierarchical assemblies of proteins exhibit a wide-range of material properties that are exploited both in nature and by artificially by humankind. Article to a diverse range of behavior including reversible softening under compression[5] and both stiffening[6] and negative normal stress under shear.[7] New insight would both further our understanding of biopolymer assemblies ubiquitous in living systems and allow for the development of biomimetic and bioinspired materials.[8−11] Recently, networks of folded globular proteins have been demonstrated to exhibit exciting cross length-scale properties,[12−15] emerging due to the added complexity and functionality of the folded building block. One approach uses so-called “fillers” to occupy the void space between the connected building blocks in the hydrogel network, restricting movement of the overall network.[22,23]
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