PROTEINS IN MOLECULAR MECHANISMS OF TEAR FILM FORMATION

这项建议的广泛的长期目标是更好地了解 泪膜润滑和保护的分子机制 人类眼表，并了解蛋白质-脂质结合 一个重要的蛋白质家族的新成员的相互作用， 脂质运载蛋白拟议的研究将有助于实现， 未来，治疗干眼症的最终目标。此外他们 可以为其他表现出广泛的脂质运载蛋白的研究提供模型。 特异性以及描述与分子重要差异 它们的特异性范围很窄 具体目标是 旨在检验泪液脂质运载蛋白在泪液分泌中发挥作用的假设。 泪膜表面脂质层的形成。此外 具体的目标是阐明所涉及的分子机制 脂质与脂质运载蛋白的结合。 具体目的I：阐明脂质运载蛋白在形成脂质体中的作用。 撕裂表面膜。 A.表面张力的贡献， 脂质运载蛋白将在水性介质中测量。泪液脂质运载蛋白 引入到含水亚相中，并且表面张力将 测定了这种蛋白质形成表面脂质和/或 将评估蛋白单层。 具体目标二：确定 泪液脂质运载蛋白作为脂质配体。 A.脂质运载蛋白对各种脂质的结合亲和力的测量 配体。竞争性结合研究将使用一系列 天然泪液中的脂质化合物。传统方法以及 采用电子顺磁共振新方法测定 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的配体相互作用的性质。的 泪液脂质运载蛋白上脂肪酸分子的结合位点的数量 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：合成的氮氧化物标记的不同碳的脂质 长度将用于测量腔的长度。的程度 泪液脂质运载蛋白腔的柔韧性将用一系列 具有大环结构的自旋标记脂质。 具体目标III：探索宽配体的分子基础 脂质运载蛋白的选择性。 分子柔性的贡献 在配体结合期间，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 在结合期间将研究多肽链的一部分。的 将研究多肽结构域在配体结合中的相互作用。 这些目标与国家咨询委员会的既定目标相一致 眼科理事会，角膜疾病子项目。

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.