Protein mechanics and engineering at the single molecule level

弹性蛋白是细胞内各种机械力化学机械中的关键弹性元件，也可以作为具有优异机械性能的结构材料。了解弹性体蛋白质的分子设计是了解各种生物过程背后的生物物理原理以及使用这些蛋白质作为自下而上构建先进材料和纳米机械设备的构建模块的关键。我们建议将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生物物理技术相结合，以阐明蛋白质机械稳定性的理化原理，开发合理的方法来设计具有可调机械性能的蛋白质，并设计新型串联模块化蛋白质基生物材料。我们将把研究重点放在小蛋白质 GB1 上，它是我们为单一蛋白质力学研究筛选的成功模型蛋白质之一。我们将使用单分子 AFM 在单分子水平上测量蛋白质的机械特性，并使用定点诱变来剖析机械展开途径，并检查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增强 GB1 机械稳定性方面取得的初步成功，我们努力将工程金属螯合方法发展成为一种通用且稳健的方法，以合理且可逆的方式调节多种蛋白质的机械稳定性。基于这些充分表征的弹性体蛋白，我们计划设计基于弹性体蛋白的生物材料，例如化学交联的水凝胶，以利用工程化到宏观生物材料中的单个弹性体蛋白中的机械特性。这些实验工作将成为使用人造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.