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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29EY011224-04
Application #
2872373
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1996-02-01
Project End
2001-01-31
Budget Start
1999-02-01
Budget End
2000-01-31
Support Year
4
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Pathology
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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
Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J (2014) Probing tertiary structure of proteins using single Trp mutations with circular dichroism at low temperature. J Phys Chem B 118:986-95

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