Protein Mechanics and Engineering at the Single Molecule Level

单分子水平的蛋白质力学和工程

• 批准号: RGPIN-2020-06024
• 负责人: Li, Hongbin
• 金额: $ 5.76万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718162
• 关键词: Protein; Mechanics; Engineering; Single; Molecule

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.

蛋白质具有纳米力学性质，这些性质独特地适合于它们在不同长度尺度上的生物学功能，从单个分子到生物体。在活细胞中，蛋白质是感受、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。了解这些机械蛋白是如何设计来执行其机械功能的，这不仅对阐明各种生物过程的基本生物物理原理很重要，而且还可以为利用蛋白质作为自下而上构建具有定制机械性能的功能生物材料的构建模块铺平道路，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）开发基于力谱的使能技术，以应对生命科学和材料科学的挑战; 2）通过编程分子序列，合理定制基于蛋白质的生物材料的机械性能，从而在单分子水平上弹性蛋白质的纳米机械性能。在这里，我们提出将联合收割机单分子原子力显微镜（AFM）、光镊与蛋白质工程以及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，这些传感器被完全校准并报告单个蛋白质在机械生物学过程中所经历的力，并利用蛋白质折叠作为驱动力来设计可以产生机械功的蛋白质生物材料。基于我们最近在AFM中实现的监测蛋白质折叠-展开接近平衡的技术进步，我们将使用单分子AFM来探测两种小金属蛋白rubredoxin和ferredoxin的折叠机制，这是使用传统方法研究的挑战。这些研究将使我们能够直接监测这些金属蛋白的折叠在真实的时间，并严格检查金属在金属蛋白的折叠中所起的作用。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要，我们将开发基于蛋白质去折叠的新一代蛋白质力传感器。我们将设计一系列具有不同且定义明确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，我们将使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致动，并设计基于蛋白质的致动器，用于机械生物学和材料科学。