PROTEINS IN MOLECULAR MECHANISMS OF TEAR FILM FORMATION

泪膜形成分子机制中的蛋白质

• 批准号: 2872373
• 负责人: BEN J GLASGOW
• 金额: $ 10.98万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2872373
• 关键词: binding proteins; chemical binding; electron spin resonance spectroscopy; gel filtration chromatography; high performance liquid chromatography; human subject; lipid structure; molecular cloning; molecular film; polymerase chain reaction; protein purification; protein structure function; site directed mutagenesis; tear; thin layer chromatography

The broad long-term objectives of this proposal are to understand better the molecular mechanisms by which the tear film lubricates and protects the human ocular surface and to understand the protein-lipid binding interactions of a new member of an important family of proteins, the lipocalins. The proposed studies will be useful in achieving, in the future, the ultimate goal of treating dry eye diseases. In addition, they may provide a model for the study of other lipocalins that exhibit broad specificity as well as delineate important differences with molecules that exhibit a narrow range of specificity. The specific aims are designed to test the hypothesis that tear lipocalins play a role in the formation of the surface lipid layer of the tear film. In addition the specific aims are designed to elucidate the molecular mechanisms involved in lipid binding to the lipocalins. Specific Aim I: To elucidate the role of lipocalins in formation of the tear surface film. A. The surface tension contribution of dilapidated and lipidated lipocalins will be measured in aqueous media. Tear lipocalins will be introduced into an aqueous subphase and the surface tension will be measured. The ability of this protein to form a surface lipid and/or protein monolayer will be assessed. Specific Aim II: To characterize the nature of the broad specificity of tear lipocalins for lipid ligands. A. Measurement of the binding affinity of lipocalins for various lipid ligands. Competitive binding studies will be performed with an array of lipid compounds found in native tears. Traditional methods as well as a novel method by electron paramagnetic resonance will be used to determine the relative affinity of lipocalins for native lipid molecules. B. Determine the nature of ligand interaction of tear lipocalins. The number of binding sites on tear lipocalin for fatty acid molecules will be determined. Competitive binding studies will be performed to explore the possibility of multiple binding sites. C. Measurement of the internal dimensions of the hydrophobic cavity in tear lipocalins: Synthetic nitroxide labeled lipids of varying carbon lengths will be used to gauge the length of the cavity. The extent of flexibility of the tear lipocalin cavity will be probed with an array of spin labeled lipids with bulky ring structures. Specific Aim III: To explore the molecular basis for the broad ligand selectivity of lipocalins. The contribution of molecular flexibility during ligand binding will be assessed by probing movement of the polypeptide backbone chain of tear lipocalins. Motion of the different portion of the polypeptide chain will be studied during binding. The interaction of polypeptide domains in ligand binding will be studied. These aims coincide with the stated objectives of the National Advisory Eye Council, corneal disease subprogram.

该提案的广泛长期目标是更好地理解 泪膜润滑和保护的分子机制 人眼表面并了解蛋白质-脂质结合 一个重要蛋白质家族新成员的相互作用 脂质运载蛋白。拟议的研究将有助于实现 未来，治疗干眼病的最终目标。此外，他们 可能为研究其他表现出广泛的脂质运载蛋白提供模型 特异性以及描述与分子的重要差异 表现出狭窄范围的特异性。 具体目标是 旨在检验泪液脂质运载蛋白在泪液中发挥作用的假设 泪膜表面脂质层的形成。此外 具体目标旨在阐明所涉及的分子机制 脂质与脂质运载蛋白的结合。 具体目标 I：阐明脂质运载蛋白在形成 撕开表面薄膜。 A. 破旧和脂化的表面张力贡献 脂质运载蛋白将在水介质中测量。泪液脂质运载蛋白将 引入到水性亚相中，表面张力将为 测量。该蛋白质形成表面脂质和/或的能力 将评估蛋白质单层。 具体目标 II：描述广泛特异性的本质 泪液脂质运载蛋白为脂质配体。 A. 脂质运载蛋白对各种脂质的结合亲和力的测量 配体。竞争性结合研究将通过一系列 天然眼泪中发现的脂质化合物。传统方法以及 将使用电子顺磁共振的新方法来确定 脂质运载蛋白对天然脂质分子的相对亲和力。 B. 确定泪液脂质运载蛋白配体相互作用的性质。这 泪脂运载蛋白上脂肪酸分子的结合位点数量 被确定。将进行竞争性结合研究以探索 多个结合位点的可能性。 C. 疏水腔内部尺寸的测量 泪液脂质运载蛋白：不同碳的合成硝基氧标记脂质 长度将用于测量空腔的长度。范围 泪液脂质运载蛋白腔的灵活性将通过一系列 具有大环结构的自旋标记脂质。 具体目标 III：探索广泛配体的分子基础 脂质运载蛋白的选择性。 分子柔性的贡献 在配体结合过程中，将通过探测配体的运动来评估 泪液脂质运载蛋白的多肽主链。不同的运动 将在结合过程中研究多肽链的一部分。这 将研究配体结合中多肽结构域的相互作用。 这些目标与国家咨询的既定目标一致 眼理事会，角膜疾病子计划。