Protein Mechanics and Engineering at the Single Molecule Level

从单分子到生物体，蛋白质具有独特的纳米机械特性，适合在不同长度的尺度上发挥生物功能。在活细胞中，蛋白质是感知、产生和承受机械力的基本工作元件，机械力被认为是调节细胞行为的重要物理线索。了解这些机械蛋白质是如何设计来执行其机械功能的，不仅对于阐明各种生物过程所依据的基本生物物理原理很重要，而且还可能为利用蛋白质作为构建自下而上构建具有定制机械性能的功能生物材料的方法铺平道路，这些材料应用于材料科学和生物医学工程。我们的研究将集中在单分子水平上的蛋白质力学和工程研究。我们研究计划的长期目标是：1)开发基于力光谱的技术，以应对生命科学和材料科学中的挑战；以及2)通过在单分子水平上对弹性蛋白质的分子序列进行编程，从而合理地定制基于蛋白质的生物材料的机械性能。在这里，我们建议将单分子原子力显微镜(AFM)、光钳与蛋白质工程和传统的生物物理技术相结合，在单分子水平上阐明金属蛋白质的完整折叠机制，开发新一代基于蛋白质的力传感器，这种传感器经过充分校准，可以报告单个蛋白质在机械生物学过程中所经历的力，并以蛋白质折叠为驱动力来设计能够产生机械功的蛋白质生物材料。在原子力显微镜监测蛋白质近平衡折叠-展开的最新技术进展的基础上，我们将利用单分子原子力显微镜来探索传统方法难以研究的两种小金属蛋白鲁布还蛋白和铁氧还蛋白的折叠机制。这些研究将使我们能够直接实时监测这些金属蛋白的折叠，并批判性地检查金属在金属蛋白折叠中所起的作用。这些研究将有助于阐明这两种金属蛋白的详细折叠机制。为了满足机械生物学和材料科学的需求，我们将开发基于蛋白质展开的新一代蛋白质力传感器。我们将设计一系列具有不同和定义良好的展开力的力传感器蛋白质，并使用它们来精确量化单个蛋白质在机械生物学过程中所经历的力。此外，我们将利用钙触发的蛋白质折叠作为一种新的机制，在单分子和宏观水平上产生机械功和驱动，并设计基于蛋白质的执行器，用于机械生物学和材料科学。

Proteins possess nanomechanical properties that are uniquely suitable for their biological functions across different length scales, from single molecules to organisms. In living cells, proteins serve as basic working elements to sense, generate and bear mechanical forces, which are recognized as important physical cues in regulating cellular behaviors. Understanding how such mechano-proteins are designed to perform their mechanical functions not only important in elucidating fundamental biophysical principles underlying various biological processes, but may also pave ways to exploiting proteins as building blocks for the bottom-up construction of functional biomaterials with tailored mechanical properties for applications in material sciences and biomedical engineering. Our research will focus on the study of protein mechanics and engineering at the single molecule level. The long term goals of our research program are: 1) to develop force spectroscopy-based enabling technologies to address challenges in life sciences and material sciences; and 2) to rationally tailor mechanical properties of protein-based biomaterials by programming the molecular sequence, and thus nanomechanical properties, of elastomeric proteins at the single-molecule level. Here we propose to combine single molecule atomic force microscopy (AFM), optical tweezers with protein engineering, and traditional biophysical techniques to elucidate the complete folding mechanism of metalloproteins at single molecule level, develop new generation of protein-based force sensors that are fully calibrated and report the force experienced by individual proteins during mechanobiological processes, and to use protein folding as a driving force to engineer protein biomaterials that can generate mechanical work. Building upon the recent technical advance we achieved in AFM to monitor protein folding-unfolding near equilibrium, we will use single molecule AFM to probe the folding mechanisms of two small metalloproteins rubredoxin and ferredoxin, which are challenging to study using traditional methods. These studies will allow us to directly monitor the folding of these metalloproteins in real time, and critically examine the role played by metal in the folding of metalloproteins. These studies will help elucidate the detailed folding mechanisms of both metalloproteins. To meet the demands of mechanobiology and materials sciences, we will develop the new generation of protein force sensors based on protein unfolding. We will engineer a series of force sensor proteins with different and well-defined unfolding forces and use them to precisely quantify the force experienced by individual proteins during mechanobiological processes. Moreover, we will use calcium-triggered protein folding as a novel mechanism to generate mechanical work and actuation at both single molecule and macroscopic levels, and engineer protein-based actuators for applications in mechanobiology and material sciences.