Micromechanics of the Extracellular Matrix

细胞外基质是由异质材料组成的预应力力学网络。 模组蛋白质和低聚糖的混合物，构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。一个 细胞外基质的特征是其组成分子在 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，用拉伸力进行响应和平衡。学习机械 这些蛋白质的活性将提供对它们在健康和 疾病缠身的国家。 组成细胞外基质蛋白的蛋白质模块的单分子研究，如 Tenascin和FN已经测量了它们的机械稳定性，显示出很强的展开层级 在他们的机械设计中。然而，仅有对展开的阻力还不足以描述 这些蛋白质必须在拉力的作用下保持机械平衡。这种平衡涉及到一个 折叠和展开运动在拉力作用下的动态平衡。分子 这种动态平衡背后的机制尚不清楚。 我们将利用单分子力谱的最新进展，这些进展现在允许 对蛋白质在恒定拉力作用下折叠和解折叠动力学的详细观察 机械力。力钳光谱学将与蛋白质工程相结合用于研究 纤维连接蛋白III型区域10FNIII的细胞结合模块的展开动力学 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 研究工程二硫键机械强度的技术及其对机械性能的影响 从蛋白质纤维蛋白和纤维连接蛋白中选择的模块的折叠/展开途径。我们的研究 将揭示蛋白质通过拉力实现动态平衡的分子机制 力，更广泛地说，展示了一种研究蛋白质折叠机制的新方法。

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.