PROTEINS IN MOLECULAR MECHANISMS OF TEAR FILM FORMATION

描述（改编自申请人摘要）：泪膜由 蛋白质、脂质和粘蛋白成分的复杂混合物， 保护人的眼表。本申请的长期目标是 是为了更好地理解蛋白质成分的分子机制， 人类的眼泪本应用程序侧重于结构-功能关系 泪液脂质运载蛋白（TL）是泪液中主要的脂质结合蛋白。的 知识的要求和机制的正常组成部分， 泪膜将有助于实现治疗干眼的最终目标 疾病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 在这个实验室里，包括氨基酸的顺序替换， 色氨酸提供有关溶液结构和主链运动的信息 蛋白质的实时分辨率在纳秒范围内。这 该应用程序旨在利用和推进这项技术， 实现以下具体目标：1）为了测试撕裂的假设， 脂质运载蛋白从角膜表面清除并溶解脂质; 2） 研究泪液脂质运载蛋白中脂质结合的分子机制。的 假设色氨酸17和异亮氨酸98有助于链 将测试形成用于脂质结合的疏水簇的相互作用。第三章 为了确定D、E和F链的二级结构， 脂质运载蛋白在溶液中; 4）为了确定结构构型， 配体特异性假设E链和F链之间的环 作为配体进入泪液脂质结合核心的pH依赖性门 将测试脂质运载蛋白。为了设计合理的治疗策略 包括用于干眼病的药物溶液， 了解泪液正常功能的分子机制 薄膜组件。预计该项目将为此作出贡献。 认识

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.