Protein Mechanics and Engineering at the Single Molecule Level

弹性蛋白是生命系统中广泛的机械化学机械中的关键弹性元件，也是具有优异机械性能的结构材料。了解此类弹性蛋白如何设计在力下发挥作用不仅对于阐明各种生物过程的基本生物物理原理很重要，而且还可以为使用这些蛋白质自下而上构建先进材料以应用于材料科学和生物医学工程铺平道路。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，以量化生物材料中单个蛋白质所经历的拉伸力，设计新型智能弹性蛋白并用它们构建基于蛋白质的动态生物材料，其机械性能可以利用外部刺激动态调整，并开发基于新型力谱的​​策略来阐明打结拓扑蛋白质的折叠机制。具体来说，我们将设计一个基于蛋白质的荧光力传感器库，可用于光学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，以设计具有所需机械性能的生物材料，用于特定的生物医学应用。利用配体结合诱导的折叠作为控制本质上无序蛋白质的折叠和展开状态的手段，我们将开发一种通用方法来设计动态蛋白质生物材料，该材料可以响应环境刺激而改变其机械性能。这种基于蛋白质的动态生物材料可能为使用蛋白质水凝胶作为生物结构模型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-展开机制，并阐明打结蛋白克服拓扑障碍折叠成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.