Folded protein hydrogel characterisation

折叠蛋白质水凝胶由交联在一起的蛋白质结构域组成，形成网络。这些反应灵敏的功能性材料利用了蛋白质的力学和生物学功能。力的施加可以破坏二级分子内键和相互作用，导致蛋白质解折叠。在网络形成期间控制蛋白质展开使得能够设计具有不同结构和机械性质的生物材料，例如，作为药物递送系统。水凝胶的结构和力学特征，通过多尺度的方法，包括原子力显微镜（AFM），小角X射线散射（SAXS），小角中子散射（SANS），和流变学。它们具有异质结构，由折叠蛋白质簇区域和未折叠蛋白质区域连接组成。小角度散射技术需要拟合模型的倒数或散射（Q-）空间数据提供间接的措施。电子显微镜（EM）技术可以提供这些结构的直接成像。然而，在EM所需的高真空条件下的脱水必须通过保持水合样品冷冻（冷冻EM）来避免。为此，制备牛血清白蛋白（BSA）水凝胶，以使用低温扫描电子显微镜（cryo-SEM）对折叠的蛋白质水凝胶进行初始成像。选择BSA是因为它是一种具有成本效益的简单球形蛋白。我们表明，低温扫描电子显微镜可以成功地用于图像的水凝胶在微米和介观尺度的结构。通过制备非常薄的样品，透射电子显微镜（TEM）将用于直接成像和探索蛋白质簇及其相互连接。

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.