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

虽然蛋白质-金属配位键通常在生物化学中发挥作用，如催化、气体运输和光合作用，但最近被证明在某些生物材料中也具有承载交联键的功能。在贻贝和海洋蠕虫、昆虫和蜘蛛的颌骨等结构中，人们发现蛋白质-金属交联物有助于材料的适应性行为，包括增加韧性和硬度、水下粘附性和自我修复；然而，结构-性质关系的细节仍未得到充分表征。我们的目标是通过使用重组表达的蛋白质作为设计金属结合位点的平台进行全面的体外研究，来探索金属-蛋白质交联物能够对如此广泛的材料行为做出贡献的机制。作为拟议研究中的模型系统，我们将使用一种名为resilin的蛋白质，它可以在体外交联并组装成宏观可扩展材料。重组表达的resilin是一个成熟的从分子到材料的系统，我们建议将能够与金属结合的组氨酸残基引入到天然序列中，或者将短的金属结合域融合到resilin结构域。在表达和纯化之后，将采用成熟的方案从含有组氨酸的resilin衍生蛋白中创建交联型生物聚合物。随后，我们将对所获得的蛋白质和交联生物材料的结构、化学和机械性能进行表征，并将通过调整引入的金属结合部位的数量和金属结合结构域的初级序列，以及通过检查不同金属离子(例如，锌、铜、镍、钴)的作用来研究机械性能的可调性。我们假设，在resilin序列中引入蛋白质金属交联点将增加通常柔软和有弹性的重组resilin的刚性、断裂应变能和滞后，但其大小将取决于蛋白质序列(例如组氨酸的数量和位置)和所使用的金属离子的类型。这项研究的结果将使我们更深入地理解具有机械功能的金属结合蛋白进化的基本原理，这可能会激发具有特殊机械性能的仿生材料。

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.