PROTEINS IN MOLECULAR MECHANISMS OF TEAR FILM FORMATION

描述(根据申请者的摘要改编)：泪膜由一个 蛋白质、脂肪和粘蛋白成分的复杂混合物，可润滑和 保护人类的眼表。此应用程序的长期目标是 是为了更好地理解蛋白质组分的分子机制 人类的眼泪。这个应用程序侧重于结构-功能关系 泪液中主要的脂结合蛋白--泪液脂质结合蛋白(TL)。这个 关于标准组件的要求和机制的知识 泪膜将有助于达到治疗干眼症的最终目标。 疾病。该实验方法利用了最近的 监测脂质结合和阐明蛋白质结构的方法 包括电子顺磁共振(EPR)、定点自旋标记、 和定点色氨酸荧光(SDTF)。SDTF是最近开发的 在这个实验室中，涉及到氨基酸的顺序替换 色氨酸提供有关溶液结构和主干运动的信息 实时分辨率在纳秒范围内的蛋白质。这 应用程序旨在利用和推动这项技术在 实现以下具体目标：1)检验撕裂的假设 Lipocalin清除和溶解角膜表面的脂类；2) 探讨泪液Lipocalin中脂质结合的分子机制。这个 色氨酸17和异亮氨酸98参与链形成的假说 将测试形成用于脂质结合的疏水簇的相互作用。3) 确定撕裂的D、E和F链的二级结构 Lipocalin在溶液中；4)确定赋予 配基专一性。假设E和F之间的环链 作为一个pH依赖的门，使配体进入泪液的脂质结合核心 将对Lipocalin进行测试。为了设计合理的治疗策略 包括干眼病的药理解决方案，必须 了解泪液正常功能的分子机制 胶片成分。预计该项目将对此作出贡献 理解。

DESCRIPTION (Adapted from applicant's abstract): The tear film is composed of a complex mixture of protein, lipid and mucin components that lubricate and protect the human ocular surface. The long term objective of this application is to understand better the molecular mechanisms of the protein components in human tears. This application focuses on the structure-function relationships of tear lipocalin (TL), the principal lipid binding protein in tears. The knowledge of the requirements and mechanisms of the normal components of the tear film will be useful in achieving the ultimate goal of treating dry eye diseases. The experimental approach takes advantage of a combination of recent methods for monitoring lipid binding and elucidating protein structure including electron paramagnetic resonance (EPR), site directed spin labeling, and site-directed tryptophan fluorescence (SDTF). SDTF was recently developed in this laboratory and involves the sequential replacement of amino acids with tryptophan to provide information about solution structure and backbone motion of proteins with a real-time resolution in the nanosecond range. This application is designed to capitalize on and advance this technology in accomplishing the following Specific Aims: 1) To test the hypothesis that tear lipocalin scavenges and solubilizes lipids from the corneal surface; 2) To investigate the molecular mechanisms of lipid binding in tear lipocalin. The hypothesis that tryptophan 17 and isoleucine 98 contribute to strand interactions to form a hydrophobic cluster for lipid binding will be tested. 3) To determine the secondary structure of the D, E, and F strands of tear lipocalin in solution; 4) To determine structural configurations that confer ligand specificity. The hypothesis that the loop between the E and F strands acts as a pH dependent gate for ligand access to the lipid binding core of tear lipocalin will be tested. In order to design logical treatment strategies including pharmacological solutions for dry eye disease, it is imperative to understand the molecular mechanisms involved in the normal function of tear film components. This project is anticipated to contribute to this understanding.