Protein mechanics and engineering at the single molecule level

弹性体蛋白质是细胞中广泛的机械力-化学机械中的关键弹性元件，也可以作为具有优异力学性能的结构材料。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并将这些蛋白质用作自下而上构建先进材料和纳米机械设备的基础的关键。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的生物物理技术相结合，以阐明蛋白质机械稳定性的物理化学原理，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生物材料。我们将重点研究一种小蛋白GB1，这是我们为单蛋白质力学研究筛选出的一种成功的模型蛋白。我们将使用单分子原子力显微镜在单分子水平上测量蛋白质的力学性质，并使用定点突变来解剖机械展开途径，并研究非共价相互作用在决定蛋白质机械稳定性中的作用。在我们利用工程金属螯合来提高GB1的机械稳定性的初步成功的基础上，我们努力将工程金属螯合方法发展成为一种以合理和可逆的方式调节不同蛋白质的机械稳定性的通用和稳健的方法。基于这些具有良好特性的弹性蛋白，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个别弹性蛋白的机械性能。这些实验工作将是将人造弹性体蛋白质用作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.