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.
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