Protein mechanics and engineering at the single molecule level

弹性蛋白质是细胞中广泛的机械化学机制中的关键弹性元件，并且还可以用作具有优异机械性能的结构材料。了解弹性蛋白质的分子设计是理解各种生物过程的生物物理原理的关键，并将这些蛋白质用作先进材料和纳米机械设备自下而上构建的构建块。我们建议联合收割机单分子原子力显微镜与蛋白质工程，分子动力学模拟和传统的生物物理技术，阐明的物理化学原理的机械稳定性的蛋白质，开发合理的方法来设计蛋白质与可调的机械性能和工程师的新型串联模块蛋白质为基础的生物材料。我们将把我们的研究重点放在一个小的蛋白质GB1，我们筛选的单蛋白质力学研究的成功模式蛋白之一。我们将使用单分子AFM在单分子水平上测量蛋白质的机械性质，并使用定点诱变来剖析机械解折叠途径，并研究非共价相互作用在确定蛋白质机械稳定性中的作用。基于我们在使用工程金属螯合来增强GB1的机械稳定性方面的初步成功，我们奋进将工程金属螯合方法发展成一种通用且稳健的方法，用于以合理且可逆的方式调节不同蛋白质的机械稳定性。基于这些充分表征的弹性蛋白，我们计划设计基于弹性蛋白的生物材料，如化学交联水凝胶，以利用宏观生物材料中设计成单个弹性蛋白的机械性能。这些实验工作将作为使用人工弹性蛋白作为各种纳米机械和材料科学应用的结构和功能构建块的第一步。

Elastomeric proteins are key elastic elements in a wide range of mechano-chemical machinery in cells, and can also function as structural materials of superb mechanical properties. Understanding the molecular design of elastomeric proteins is the key to understand the biophysical principles underlying various biological processes and to use these proteins as building blocks for the bottom-up construction of advanced materials and nanomechanical devices. We propose to combine single molecule atomic force microscopy with protein engineering, molecular dynamics simulations and traditional biophysical techniques to elucidate the physiochemical principles underlying the mechanical stability of proteins, to develop rational methodologies to design proteins with tunable mechanical properties and to engineer novel tandem-modular protein based biomaterials. We will focus our studies on a small protein GB1, one of the successful model proteins we screened for single protein mechanics studies. We will use single molecule AFM to measure the mechanical properties of proteins at the single molecule level and use site-directed mutagenesis to dissect the mechanical unfolding pathways and examine the roles of non-covalent interactions in determining the mechanical stability of proteins. Building upon our initial success in using engineered metal chelation to enhance the mechanical stability of GB1, we endeavor to develop the engineered metal chelation approach into a general and robust methodology for modulating the mechanical stability of diverse proteins in a rational and reversible fashion. Based on these well-characterized elastomeric proteins, we plan to engineer elastomeric protein-based biomaterials, such as chemically crosslinked hydrogels, to harness the mechanical properties engineered into individual elastomeric proteins in macroscopic biomaterials. These experimental efforts will serve as the first step towards using artificial elastomeric proteins as structural and functional building blocks for a variety of nanomechanical and material science applications.