ESR SPIN LABEL STUDIES OF FIBRONECTINS

纤连蛋白的 ESR 旋转标签研究

• 批准号: 3288813
• 负责人: CHING-SAN LAI
• 金额: $ 13.31万
• 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/3288813
• 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.

纤连蛋白是血液和组织的主要蛋白质，其在血液和组织中起着中枢调节作用。 细胞粘附的作用。 GM35719的目标是获得更深层次的 了解各区域的三维关系， 纤连蛋白，以及配体结合和分子结合的结构基础 扩张. 本更新申请的具体目的1是表征 等离子体各功能区之间的动态相互作用 使用电子自旋共振（ESR）光谱法测定纤维连接蛋白。 过场 在各种条件下的纤连蛋白分子及其片段将 通过估计有效旋转相关时间来研究， 用计算机模拟实验光谱。 具体目标二是 为了确定纤连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 新的限制，以完善目前的三维模型， 纤连蛋白结构 具体目标3是研究非共价键 通过使用单体纤连蛋白的纤连蛋白二聚体的缔合。 的 接触位点和两者之间非共价缔合的性质 缺乏链间二硫键的纤连蛋白单体， 通过使用ESR和荧光光谱进行研究。 具体目标 4是表征重组纤连蛋白片段表达在 利用分子克隆技术的细菌系统。 定点 通过取代链间半胱氨酸残基的诱变 与丝氨酸残基的二硫键将用于确定 无论是羧基端的两个链间二硫键， 分子以平行或反平行方式排列。 具体 目的5是研究重组体的结构和动力学 使用定点诱变和 生物物理技术 纤连蛋白片段将在基因上 设计成包含单个选择性标记位点，一次一个， 而不影响蛋白质的功能。 这使得自旋的位置 标记或荧光标记插入重组纤连蛋白片段中， 通过ESR阐明详细的结构信息，或 荧光分析 这些研究将提供以前 无法获得有关三维结构的信息， 纤连蛋白分子和机制，通过其功能解蔽 某些结构域响应于配体诱导的构象变化而发生， 改变。 这些信息应该为解释 纤连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。