Mechanical tunability of biological materials via protein-metal cross-linking

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Although typically known to function in biochemical roles such as catalysis, gas transport, and photosynthesis, protein-metal coordination bonds were recently demonstrated to also function as load-bearing cross-links in certain biological materials. In structures such as the mussel byssus and the jaws of marine worms, insects and spiders, it was found that protein-metal cross-links contribute to adaptive material behaviors including increased toughness and hardness, underwater adhesion, and self-repair; however, the details of the structure-property relationships remain poorly characterized. We aim here to explore the mechanisms by which metal-protein cross-links are able to contribute to such a wide range of material behaviors by undertaking a comprehensive in vitro investigation using recombinantly expressed proteins as a platform in which to engineer metal-binding sites. As a model system in the proposed study, we will use a protein called resilin, which can be cross-linked in vitro and assembled into a macroscale extensible material. Recombinantly expressed resilin is a well-established system for going from molecules to materials, and we propose to introduce histidine residues capable of metal binding into the native sequence or, alternatively, to fuse short metal binding domains to resilin domains. Following expression and purification, well-established protocols will be employed to create cross-linked biopolymers from the histidine-containing resilin-derived proteins. We will subsequently characterize the structural, chemical and mechanical properties of the obtained proteins and cross-linked biomaterials and will investigate the tunability of mechanical properties by adjusting the number of introduced metal-binding sites and the primary sequence of the metal-binding domains, as well as by examining the role of different metal ions (e.g. Zn2+, Cu2+, Ni2+, Co2+). We hypothesize that introducing protein metal cross-linking sites into the resilin sequence will increase the stiffness, strain energy to break and hysteresis exhibited by the normally soft and resilient recombinant resilin, but the magnitude will be dependent on protein sequence (e.g. number and position of histidines) and the types of metal ions employed. The result of this study will provide a deeper understanding of the fundamental principles underlying the evolution of metal binding proteins with mechanical functions, which may have potential to inspire biomimetic materials with exceptional mechanical properties.