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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011224-09
Application #
6705054
Study Section
Visual Sciences A Study Section (VISA)
Program Officer
Fisher, Richard S
Project Start
1996-02-01
Project End
2006-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
9
Fiscal Year
2004
Total Cost
$305,000
Indirect Cost
Name
University of California Los Angeles
Department
Pathology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Glasgow, Ben J; McCannel, Tara A (2018) Correlation of Immunocytochemistry of BRCA1-associated Protein-1 (BAP1) With Other Prognostic Markers in Uveal Melanoma. Am J Ophthalmol 189:122-126
Glasgow, Ben J; Abduragimov, Adil R (2018) Interaction of ceramides and tear lipocalin. Biochim Biophys Acta Mol Cell Biol Lipids 1863:399-408
Glasgow, Ben J; Abduragimov, Adil R (2018) Ligand binding studies by high speed centrifugal precipitation and linear spectral summation using ultraviolet-visible absorption spectroscopy. MethodsX 5:345-351
Glasgow, Ben J; Abduragimov, Adil R (2018) Data on Orphan tear lipid analogs, synthesis and binding to tear lipocalin. Data Brief 18:999-1004
Glasgow, Ben J; Ma, Lie (2016) Simultaneous two color image capture for sub-diffraction localization fluorescence microscopy. Micron 80:14-9
Glasgow, Ben J (2016) Conventional fluorescence microscopy below the diffraction limit with simultaneous capture of two fluorophores in DNA origami. Proc SPIE Int Soc Opt Eng 9714:
Glasgow, Ben J (2016) Fluorescence lifetime imaging microscopy reveals quenching of fluorescein within corneal epithelium. Exp Eye Res 147:12-19
Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J (2015) Exploring protein solution structure: Second moments of fluorescent spectra report heterogeneity of tryptophan rotamers. Spectrochim Acta A Mol Biomol Spectrosc 150:909-20
Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J (2015) Double tryptophan exciton probe to gauge proximal side chains in proteins: augmentation at low temperature. J Phys Chem B 119:3962-8
Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J (2014) A simple model-free method for direct assessment of fluorescent ligand binding by linear spectral summation. J Fluoresc 24:231-8

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