Folded protein hydrogel characterisation

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Folded protein hydrogels consist of protein domains crosslinked together, forming a network. These responsive, functional materials exploit the protein's mechanics and biological function. The application of force can break secondary intramolecular bonds and interactions, leading to protein unfolding. Control of protein unfolding during network formation enables the design of biomaterials with distinct architecture and mechanical properties with application for example, as drug delivery systems. Hydrogel structures and mechanics have been characterised through a multiscale approach involving atomic force microscopy (AFM), small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), and rheology. They have a heterogeneous structure consisting of areas with folded protein clusters connected by areas of unfolded protein. Small angle scattering techniques require fitting models to reciprocal or scattering (Q-)space data providing indirect measures. Electron microscopy (EM) techniques could provide direct imaging of these structures. However, dehydration under the high vacuum conditions required for EM must be avoided by keeping the hydrated sample frozen (cryo-EM). For this purpose, a bovine serum albumin (BSA) hydrogel was prepared to initially image a folded protein hydrogel using cryo-scanning electron microscopy (cryo-SEM). BSA was selected because it is a cost-effective, simple globular protein. We show that cryo-SEM can be successfully used to image the structure of the hydrogels at the micrometre and mesoscale. By preparing very thin samples, transmission electron microscopy (TEM) will be used to directly image and explore the protein clusters and their interconnections.