Alpha-crystallin, the major protein component of the vertebrate eye lens, exists in situ as a heterogeneous population of aggregates averaging about 13nm. The small size of these aggregates, combined with their very high concentration and long-term stability, contribute to the increased refractive index gradient of the lens needed for image focusing on the retina, the transparency of the lens in the visible wavelength spectrum, and the longevity of lens function in vision. The protein has, however, been implicated in the development of cataracts, as the increased population of larger-sized soluble particles and insoluble material is largely alpha-crystallin. Despite alpha-crystallin's critical importance in visual function and its major role in cataractogenesis, however, surprisingly little is known about the solubility characteristics of the subunit, the aggregation process into particles, the effects of environmental shifts on aggregation state, or solution interactions of the particles at physiological concentrations. This is due in large part to the fact that alpha-crystallin aggregates cannot be dissociated without concomitant denaturation of the subunits, preventing the systematic exploration of the protein's unique properties. Hydropathy analysis of the peptide sequence, combined with analysis of literature reports, led to the formulation in 1987 by the PI and a colleague of the micelle hypothesis for alpha-crystallin, suggesting that it behaved like the protein version of an amphipathic molecule and was stabilized in aggregate form through hydrophobic interactions. This hypothesis has been tested and validated using several independent biophysical techniques, and serves as the basis for experimental strategies designed to explore the three specific aims of this proposal: differential biophysical characterization of the four alpha-crystallin isoforms; biophysical characterization of the solution behavior of alpha-crystallin aggregates below, at and above physiological concentrations; and biophysical characterization of mixed populations of the three lens crystallins below, at, and above physiological concentrations. Techniques to be employed in the accomplishment of these aims include circular dichroism spectropolarimetry, fast performance liquid chromatography, hydrostatic pressure application, rotational and oscillatory rheometry, osmotic stress, electron microscopy, and synchrotron scattering and diffraction. Development of methods to characterize the alpha-crystallin molecule alone and in aggregate form, as well as to describe its interactions in solution at physiological concentrations, is a critical prerequisite for focused development of strategies to inhibit cataractogenesis.
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