The long term objectives of this proposal are to elucidate the structural features and interactions of the crystallins that endow these proteins with stability and transparency and to understand the cause and mechanism by which structural destabilization leads to aggregation of the proteins and loss of transparency during cataractogenesis.
The Specific Aims of this project period are: 1. To test the hypothesis that at physiological concentration lens crystallins interact to undergo a conformational readjustment from the usual beta-sheet to an extended interconnected multimolecular assembly to provide short-range order and transparency. Studies include: (a) In vitro measurements of molecular interactions from dilute to physiological concentration. (b) Calorimetry to determine the thermodynamics of the process. Proteins from both bovine and human lens will be used for this study. 2. To test the hypothesis of molecular chaperone activity of alpha- crystallin and to define this function in terms of the transparency of the lens. Studies include: (a) Thermodynamics and conformational aspects of chaperone-target protein interactions using spectroscopic techniques. (b) Mechanism for chaperone-target protein interactions. 3. To determine the extent of protein modifications during cataractogenesis and to define their role in the structural destabilization during cataract formation. Studies include; (a) Extensive physico- chemical and biochemical analysis of freshly obtained human cataractous lens. (b) Determination of the level of glycation, pigmentation, glutathione, protein-protein disulfide and protein glutathione mixed disulfide and crosslink formation in cataractous lens crystallins and assessment of the role played by these modified proteins in their structural destabilization during cataract formation. In addition to appropriate chemical, biochemical and spectroscopic techniques, such as, absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FTIR), the project will also use (a) high resolution differential scanning calorimetry (DSC) (b) nuclear magnetic relaxation dispersion (NMRD) and (c) immunological techniques to accomplish the aims.
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