PROTEINS IN MOLECULAR MECHANISMS OF TEAR FILM FORMATION

这项建议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 人的眼表和了解蛋白质-脂结合 一个重要蛋白质家族的新成员之间的相互作用 脂钙蛋白。建议的研究将有助於在 未来，治疗干眼病的最终目标。此外，他们还 为研究其他具有广泛应用前景的脂钙蛋白提供了一个模型 以及描述与分子的重要区别 表现出很小范围的特异性。具体目标是 旨在测试泪液脂结合蛋白在 泪膜表面脂层的形成。此外， 设计特定的目的是为了阐明所涉及的分子机制。 在脂类与脂类结合时。 具体目的一：阐明脂钙蛋白在血管形成中的作用 撕裂面膜。 A.破旧和油化的表面张力的贡献 Lipocalin将在水介质中进行测量。眼泪脂钙蛋白将是 引入水相子相中，表面张力将被 量过了。这种蛋白质形成表面脂和/或的能力 将对蛋白质单分子层进行评估。 具体目标二：描述生物多样性广泛特殊性的性质 撕裂Lipocalin作为脂类配体。 A.Lipocalin与各种脂类结合亲和力的测定 配基。竞争性结合研究将与一系列 天然眼泪中发现的类脂化合物。传统的方法以及 将使用电子顺磁共振的新方法来确定 Lipocalin对天然脂分子的相对亲和力。 确定泪液脂联蛋白的配基相互作用的性质。这个 泪液Lipocalin与脂肪酸分子的结合位点数将 要下定决心。将进行竞争性约束性研究，以探索 多个结合位点的可能性。 C.疏水空腔内部尺寸的测量 眼泪脂钙蛋白：合成氮氧化物标记的不同碳的脂类 长度将被用来测量腔的长度。其影响范围 泪液Lipocalin腔的灵活性将通过一组 自旋标记的具有笨重环状结构的脂类。 特殊目的III：探索广谱配体的分子基础 脂钙蛋白的选择性。分子柔性的贡献 在配基结合过程中，将通过探测 泪液脂钙蛋白的多肽主链。不同的人的运动 多肽链的一部分将在结合过程中进行研究。这个 将研究配体结合过程中多肽结构域之间的相互作用。 这些目标与国家咨询委员会宣布的目标不谋而合 眼科委员会，角膜病子计划。

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.