Micromechanics of the Extracellular Matrix

细胞外基质是一种由异质性细胞组成的预应力力学网络， 构成活组织的机械支架的模块蛋白质和寡糖的混合物。它 在形成所有生物体的细胞结构的发展中起着至关重要的作用。一 细胞外基质的特征性特征是其组成分子在细胞外基质的作用下起作用。 拉伸力我们的长期目标是了解这些模块化蛋白质 在单分子水平上，与拉伸力反应并平衡。学习机械 这些蛋白质的活性将提供对它们在健康和 病态的国家 对组成细胞外基质蛋白的蛋白模块的单分子研究， 腱生蛋白和纤连蛋白已经测量了它们的机械稳定性，揭示了强烈的展开层次 在他们的机械设计中。然而，对解折叠的抗性不足以描述 这些蛋白质必须在机械上与拉力保持平衡。这种平衡涉及 折叠和展开事件的动态平衡对抗拉力发生。分子 这种动力学平衡的机制尚不清楚。 我们将利用单分子力光谱学的最新进展， 详细观察蛋白质的折叠和展开动力学，同时被一个常数拉动， 机械力力钳光谱结合蛋白质工程将用于研究 纤连蛋白III型区域的细胞结合模块10FNIII的解折叠动力学，以及 细胞外基质蛋白的其他关键模块。我们将使用该技术的强制淬火模式 研究蛋白质在拉伸力作用下的折叠机制。我们将使用单分子 技术来研究工程二硫键的机械强度及其对 来自蛋白质纤维连接蛋白和纤连蛋白的选定模块的折叠/解折叠途径。我们的研究 将揭示蛋白质与拉力动态平衡的分子机制， 力，更一般地说，展示了一种新的方法来研究蛋白质折叠的机制。

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.