Micromechanics of the Extracellular Matrix

描述(申请人提供)：细胞外基质是一种预应力机械网络，由模块蛋白质和寡糖的异质混合物组成，形成活组织的机械支架。它在构成所有生命有机体的细胞结构的发展中起着至关重要的作用。细胞外基质的一个特征是其组成分子在拉伸力作用下发挥作用。我们的长期目标是在单分子水平上了解这些模块化蛋白的反应机制，并与伸缩力保持平衡。研究这些蛋白质的机械活性将从机制上理解它们在健康和疾病状态下的功能。对构成细胞外基质蛋白的蛋白质模块(如肌腱蛋白和纤维连接蛋白)的单分子研究已经测量了它们的机械稳定性，揭示了它们机械设计中强烈的展开层级。然而，抗去折叠能力不足以描述这些蛋白质的功能，这些蛋白质必须与拉力机械地平衡。这种平衡包括折叠和展开活动与拉力之间的动态平衡。这种动态平衡背后的分子机制尚不清楚。我们将利用单分子力谱的最新进展，现在可以在恒定的机械力的拉动下，详细观察蛋白质的折叠和去折叠动力学。力钳光谱结合蛋白质工程将用于研究纤维连接蛋白III区的细胞结合模块、10FNIII以及其他细胞外基质蛋白的关键模块的去折叠动力学。我们将利用这一技术的力-猝灭模式来研究蛋白质在拉伸力下折叠的机制。我们将使用单分子技术来研究工程化二硫键的机械强度以及它们对从蛋白质纤维蛋白和纤维连接蛋白中选择的模块折叠/去折叠路径的影响。我们的研究将揭示蛋白质在拉力作用下动态平衡的分子机制，更广泛地说，将展示一种研究蛋白质折叠机制的新方法。

DESCRIPTION (provided by applicant): The extracellular matrix is a pre-stressed mechanical network composed of a heterogeneous mixture of modular proteins and oligosaccharides that form the mechanical scaffold of living tissues. It plays crucial roles in the development of the cellular architectures that form all living organisms. A characteristic feature of the extracellular matrix is that its constituent molecules function under a stretching force. Our long-term aim is to understand the mechanisms by which these modular proteins respond and equilibrate with a stretching force, at the single molecule level. Studying the mechanical activity of these proteins will provide a mechanistic understanding of their function in healthy and diseased states. Single molecule studies of the protein modules that compose extracellular matrix proteins such as tenascin and fibronectin have measured their mechanical stability, revealing strong unfolding hierarchies in their mechanical design. However, resistance to unfolding is not sufficient to describe the function of these proteins, which must equilibrate mechanically against a pulling force. This equilibration involves a dynamic balance of folding and unfolding events taking place against the pulling force. The molecular mechanisms underlying this kinetic equilibrium are unknown. We will take advantage of recent advances in single molecule force spectroscopy that now permit a detailed observation of the folding and unfolding kinetics of a protein, while being pulled by a constant mechanical force. Force-clamp spectroscopy combined with protein engineering will be used to study the dynamics of unfolding of the cell binding module of the type III region of fibronectin, 10FNIII, and other key modules of extracellular matrix proteins. We will use the force-quench mode of this technique to study the mechanisms of protein folding under a stretching force. We will use single molecule techniques to study the mechanical strength of engineered disulphide bonds and their effect on the folding/unfolding pathways of selected modules from the proteins fibrillin and fibronectin. Our studies will reveal the molecular mechanisms underlying the dynamic equilibration of proteins with a pulling force, and more generally, demonstrate a novel approach to study the mechanisms of protein folding.