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Micromechanics of the Extracellular Matrix

细胞外基质的微观力学

基本信息

批准号：
7161732
负责人：
Julio M Fernandez
金额：
$ 38.5万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-01-01 至 2009-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7161732
关键词：
Amino Acids Architecture Arsenic Behavior Binding Boat Cells Characteristics Complex Cysteine Data Development Engineering Equilibrium Event Extracellular Matrix Extracellular Matrix Proteins Fibronectins Funding Heavy Metals Heparin Kinetics Life Location Maps Measures Mechanical Stress Mechanics Memory Methods Modeling Molecular Molecular Conformation Mutagenesis Numbers Oligosaccharides Organism Pathway interactions Physiologic pulse Play Polyproteins Polysaccharides Protein Biochemistry Protein Conformation Protein Engineering Proteins Protocols documentation Pulse taking Rate Reaction Research Personnel Resistance Role Rupture Spectrum Analysis Staging Standards of Weights and Measures Stress Stretching Techniques Temperature Tenascin Thermodynamics Thinking Time Tissues Ubiquitin Work base boating design disulfide bond driving force expectation fibrillin instrumentation mutant novel novel strategies oxidation programs protein folding protein function scaffold single molecule sugar

项目摘要

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.

描述（由申请人提供）：细胞外基质是一种预应力机械网络，由形成活组织机械支架的模块蛋白和寡糖的异质混合物组成。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。细胞外基质的特征性特征是其组成分子在拉伸力下起作用。我们的长期目标是在单分子水平上了解这些模块化蛋白质响应并与拉伸力平衡的机制。研究这些蛋白质的机械活性将提供对它们在健康和疾病状态下功能的机械理解。单分子研究的蛋白质模块，组成细胞外基质蛋白，如腱生蛋白和纤连蛋白测量其机械稳定性，揭示了强大的展开层次结构，在其机械设计。然而，对解折叠的阻力不足以描述这些蛋白质的功能，这些蛋白质必须在机械上与拉力平衡。这种平衡涉及对抗拉力发生的折叠和展开事件的动态平衡。这种动力学平衡背后的分子机制是未知的。我们将利用单分子力谱的最新进展，现在允许详细观察蛋白质的折叠和展开动力学，同时被恒定的机械力拉动。结合蛋白质工程的力钳光谱将被用来研究纤连蛋白，10 FNIII，和细胞外基质蛋白的其他关键模块的III型区域的细胞结合模块的展开的动力学。我们将使用该技术的力淬灭模式来研究拉伸力下蛋白质折叠的机制。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蛋白质纤维连接蛋白和纤连蛋白中选定模块的折叠/展开途径的影响。我们的研究将揭示蛋白质与拉力动态平衡的分子机制，更普遍地说，展示了一种新的方法来研究蛋白质折叠的机制。