Protein Mechanics and Engineering at the Single Molecule Level

弹性体蛋白质是生命系统中各种机械力化学机械中的关键弹性元件，也是具有优异力学性能的结构材料。了解这些弹性体蛋白质是如何在力的作用下发挥作用的，不仅对于阐明各种生物过程背后的基本生物物理原理很重要，而且可能为使用这些蛋白质自下而上地构建先进材料在材料科学和生物医学工程中的应用铺平道路。我们建议将单分子力谱与蛋白质工程、分子动力学模拟和传统生物物理技术相结合，合理设计基于蛋白质的荧光力传感器，以量化单个蛋白质在生物材料中所经历的拉伸力，设计新型智能弹性蛋白质，并利用它们构建可根据外部刺激动态调整力学性能的蛋白质基动态生物材料，并开发基于力谱的新策略，以阐明蛋白质的打结拓扑结构的折叠机制。具体地说，我们将设计一个基于蛋白质的荧光力传感器库，可以用来光学地量化生物材料中单个蛋白质所经历的拉伸力。对这种分子尺度作用力的了解将有助于弥合单分子力学和生物材料力学之间的差距，并有助于在分子水平上定制弹性蛋白质，以设计出具有特定生物医学应用所需机械性能的生物材料。利用配体结合诱导折叠作为一种手段来控制内在无序蛋白质的折叠和去折叠状态，我们将开发一种通用的方法来设计能够响应环境刺激而改变其力学性能的动态蛋白质生物材料。这种基于蛋白质的动态生物材料可能为将蛋白质水凝胶用作模型生物结构和组织再生开辟了可能性。此外，我们还将利用单分子力谱和分子动力学模拟来阐明打结tRNA甲基转移酶TrmD的折叠-去折叠机制，并揭示打结蛋白克服拓扑障碍折叠成其复杂功能结构的机制。

Elastomeric proteins are key elastic elements in a wide range of mechano-chemical machinery in living systems, and also constitute structural materials of superb mechanical properties. Understanding how such elastomeric proteins are designed to function under force is not only important in elucidating fundamental biophysical principles underlying various biological processes, but may also pave ways to using these proteins for the bottom-up construction of advanced materials for applications in materials science and biomedical engineering. We propose to combine single molecule force spectroscopy with protein engineering, molecular dynamics simulations and traditional biophysical techniques to rationally engineer protein-based fluorogenic force sensors to quantify stretching forces experienced by individual protein in biomaterials, engineer novel smart elastomeric proteins and use them to construct protein-based dynamic biomaterials whose mechanical properties can be dynamically tuned using external stimuli, and develop novel force spectroscopy-based strategies to elucidate the folding mechanism of proteins of knotted topologies. Specifically, we will engineer a library of protein-based fluorogenic force sensors that can be used to quantify the stretching force experienced by individual protein in biomaterials optically. The knowledge of such molecular scale forces will help bridge the gap between single molecule mechanics and mechanics of biomaterials, and help tailor elastomeric proteins at the molecular level to engineer biomaterials with desirable mechanical properties for specific biomedical applications. Making use of ligand binding-induced folding as a means to control the folded and unfolded states of intrinsically disordered proteins, we will develop a general method to engineer dynamic protein biomaterials that can change their mechanical properties in response to environmental stimuli. Such protein-based dynamic biomaterials may open up possibilities for using protein hydrogels as model biological structures and in tissue regeneration. Moreover, we will also use single molecule force spectroscopy and molecular dynamics simulations to elucidate the folding-unfolding mechanism of a knotted tRNA methyltransferase TrmD, and shine light on the mechanism by which knotted proteins overcome topological barrier to fold into their complex functional structures.**