PROTEINS IN MOLECULAR MECHANISMS OF TEAR FILM FORMATION

泪膜形成分子机制中的蛋白质

• 批准号: 2716455
• 负责人: BEN J GLASGOW
• 金额: $ 10.39万
• 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/2716455
• 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 催泪膜润滑并保护的分子机制 人眼表面并了解蛋白质脂质结合 重要的蛋白质家族的新成员的相互作用， Lipocalins。拟议的研究将在实现，在 未来，治疗干眼症的最终目标。另外，他们 可以为研究其他脂肪蛋白的研究提供模型 特异性以及描述分子的重要差异 表现出狭窄的特异性。 具体目的是 旨在测试撕裂脂肪蛋白在 泪膜的表面脂质层的形成。另外 特定目的旨在阐明涉及的分子机制 在脂质结合与Lipocalins中。 特定目的I：阐明脂蛋白在形成中的作用 撕裂表面膜。 答：破旧和脂化的表面张力贡献 Lipocalins将在水性培养基中进行测量。撕裂脂肪蛋白会 引入水下，表面张力将是 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。 特定目的II：表征广泛特异性的性质 脂肪蛋白撕裂脂质蛋白。 A.测量Lipocalins对各种脂质的结合亲和力 配体。竞争性的结合研究将通过一系列 脂质化合物在天然泪液中发现。传统方法以及 电子顺磁共振的新方法将用于确定 Lipocalins对天然脂质分子的相对亲和力。 B.确定泪液lipococalins的配体相互作用的性质。这 脂肪酸分子的泪液中的结合位点的数量将 可以确定。将进行竞争性约束力研究以探索 多个结合位点的可能性。 C.测量疏水腔内内部尺寸 撕裂脂蛋白：合成氮氧化物标记为不同碳的脂质 长度将用于评估腔的长度。程度 泪液lipocalin腔的灵活性将用一系列探测 带有笨重的环结构的自旋标记脂质。 特定目标III：探索宽配体的分子基础 脂肪蛋白的选择性。 分子灵活性的贡献 在配体结合过程中，将通过探测运动的运动来评估 多肽骨链链蛋白链链链链。不同的运动 在结合过程中将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相吻合 眼科委员会，角膜疾病子程序。