ESR SPIN LABEL STUDIES OF FIBRONECTINS

纤连蛋白的 ESR 旋转标签研究

• 批准号: 3288819
• 负责人: CHING-SAN LAI
• 金额: $ 13.51万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288819
• 关键词: chemical association; circular dichroism; computer simulation; conformation; dimer; disulfide bond; electron spin resonance spectroscopy; fibronectins; fluorescence spectrometry; fluorescent dye /probe; high performance liquid chromatography; human tissue; hydropathy; intermolecular interaction; ionic strengths; molecular cloning; molecular dynamics; molecular site; mutant; protein engineering; protein purification; protein structure function; recombinant DNA; sedimentation equilibrium; site directed mutagenesis

Fibronectin is a major protein of blood and tissues that plays a central role in cell adhesion. The goal of GM35719 is to gain a deeper understanding of the three-dimensional relationship of various regions of fibronectin, and of the structural basis for ligand binding and molecular expansion. The specific aim 1 of this renewal application is to characterize the dynamic interactions between various functional domains of plasma fibronectin using electron spin resonance (ESR) spectroscopy. The motions of the fibronectin molecule and its fragments under various conditions will be studied by estimating the effective rotational correlation times from the experimental spectra using computer simulations. The specific aim 2 is to determine the intramolecular distances of the fibronectin molecule using fluorescence energy transfer techniques. These distances will be used as new constraints to refine the current three-dimensional model of fibronectin structure. The specific aim 3 is to study the non-covalent association of fibronectin dimers by using monomeric fibronectin. The contact sites and the nature of non-covalent association between the two monomers of fibronectin, which lack the interchain disulfide bonds, will be studied by using both ESR and fluorescence spectroscopy. The specific aim 4 is to characterize recombinant fibronectin fragments expressed in bacterial systems using molecular cloning techniques. Site-directed mutagenesis by substitution of a cysteine residue involved in interchain disulfide bonding with a serine residue will be employed to determine whether the two interchain disulfide bridges in the carboxyl ends of the molecule are arranged in parallel or antiparallel fashion. The specific aim 5 is to investigate the structure and dynamics of the recombinant fibronectin fragments using a combination of site-directed mutagenesis and biophysical techniques. Fibronectin fragments will be genetically engineered to contain single selective labeling sites, one at a time, without affecting the protein function. This permits the placement of spin labels or fluorescent labels into the recombinant fibronectin fragment and the elucidation of detailed structural information through ESR or fluorescence analysis. The proposed studies will provide previously unavailable information regarding the three-dimensional structure of the fibronectin molecule and mechanisms by which functional unmasking of certain domains occurs in response to ligand-induced conformational alterations. This information should provide a basis for explaining important roles of fibronectin in cell attachment and migration, embryogenesis, wound healing, and oncogenic transformation.

纤维连接蛋白是血液和组织中的一种主要蛋白质，它在 在细胞黏附中的作用。GM35719的目标是获得更深层次的 对中国各区域立体关系的认识 纤维连接蛋白，以及配体结合和分子的结构基础 扩张。 此续订申请的具体目标1是描述 血浆各功能结构域之间的动态相互作用 纤维连接蛋白的电子自旋共振(ESR)波谱分析。这些动议 纤维连接蛋白分子及其片段在不同条件下 通过估计有效的旋转相关时间来研究 用计算机模拟实验光谱。具体目标2是 用来确定纤维连接蛋白分子的分子内距离 荧光能量转移技术。这些距离将被用作 改进当前三维模型的新约束 纤维连接蛋白结构。具体目标3是研究非共价键 利用单体纤维连接蛋白结合纤维连接蛋白二聚体。这个 接触部位和两者之间非共价缔合的性质 缺少链间二硫键的纤维连接蛋白单体将是 采用电子自旋共振和荧光光谱分析相结合的方法进行研究。具体目标 4是鉴定重组纤维连接蛋白片段的表达。 使用分子克隆技术的细菌系统。站点定向 链间半胱氨酸残基的替代诱变 将使用与丝氨酸残基的二硫键来确定 羧基末端的两个链间二硫键是否 分子以平行或反平行的方式排列。具体的 目的5是研究重组蛋白的结构和动力学 使用定点突变和联合应用的纤维连接蛋白片段 生物物理技术。纤维连接蛋白片段将从基因上 设计成包含单一的选择性标记位置，一次一个， 而不影响蛋白质的功能。这允许放置旋转 将标记或荧光标记插入到重组纤维连接蛋白片段中 通过ESR或ESR阐明详细的结构信息 荧光分析。拟议的研究将提供以前的 无法获得有关三维结构的信息 纤维连接蛋白分子及其功能揭开的机制 某些结构域的出现是对配体诱导的构象的响应 改装。这些信息应该提供解释的基础 纤维连接蛋白在细胞附着和迁移中的重要作用 胚胎发生、伤口愈合和致癌转化。