The overall goal of this proposal is to learn the role of proteins in the molecular mechanisms in human tear film formation. The project has potential to significantly impact the way proteins are studied in solution. We propose to capitalize on the technologic innovations developed in our laboratory of site directed tryptophan fluorescence and circular dichroism to further this effort. In addition the project may provide the basis for protein engineering of tear lipocalin for specific eye treatments. Since tear lipocalin is decreased in dry eye, the study of its functions and mechanisms will provide a better understanding of how a major protein component of human tears functions normally and what is needed to treat dry eye disease. The proposal has 2 specific aims: 1: Elucidation of tryptophan rotamer configurations in tear lipocalin by site directed tryptophan fluorescence lifetimes. 2. To deconvolve the near UV Circular Dichroism (CD) tryptophan spectra to identify specific loop conformers in tear lipocalin.
AIM1. While amino acid rotamers are fundamental to mechanisms of protein function, they can only be deduced indirectly by nuclear magnetic resonance (NMR) and only for some proteins. Crystallography usually can assign one rotamer (the most populated one) for each amino acid residue and is limited to only the very few proteins which have exceedingly high resolution crystal structures. This proposal aims to create a direct method to observe rotamers of tryptophan in proteins in solution. While similar approaches were started in small synthetic peptides, success is lacking in complete natural proteins. We propose to use the backbone restraints of tear lipocalin combined our expertise in time resolved site directed tryptophan fluorescence to resolve rotamer populations in lifetime fluorescence. Success will provide a needed way to probe backbone and amino acid side chain constraints of rotamers in all proteins.
AIM 2. We will test the hypothesis that low temperature UV CD tryptophan spectra will resolve conformational movement of the AB loop of tear lipocalin. The flexible loop regions in proteins, such as the loop AB of tear lipocalin, are critical to ligand binding but can not be resolved in most crystal structures. NMR is limited to indirect information and only in some proteins. By combining low temperature near UV site directed tryptophan CD and novel multi-variant spectral deconvolution software the vibronic structure of the loop can be for the first time educed. Generated will be an important tool for identifying loop conformations that are critical to ligand binding to aid our understanding of the critical loop function of tear lipocalin and many other proteins. The information is critical to engineering proteins to treat general medical as well as eye diseases.
of this project is to learn the intricate molecular interactions of proteins in human tears. The goal is to develop new technologies to interrogate the molecular mechanisms in tears and at the same time learn how tear proteins function in health and disease.
|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 147:12-9|
|Glasgow, Ben J; Ma, Lie (2016) Simultaneous two color image capture for sub-diffraction localization fluorescence microscopy. Micron 80:14-9|
|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 (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 (2014) Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin. Biochem Biophys Res Commun 452:1004-8|
|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|
|Staudinger, Tamara; Redl, Bernhard; Glasgow, Ben J (2014) Antibacterial activity of rifamycins for M. smegmatis with comparison of oxidation and binding to tear lipocalin. Biochim Biophys Acta 1844:750-8|
|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 (2013) Effect of short- and long-range interactions on trp rotamer populations determined by site-directed tryptophan fluorescence of tear lipocalin. PLoS One 8:e78754|
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