Alpha-crystallin, tha major protein component of the crystalline lens of mammalian eyes , exist in the lens cytoplasm as aggregates of approximately 40 subunits in an isoform mixture of 3A:1B in the human. Because of the way the lens develops throughout the lifetime of an organism, a-crystallin and other lens proteins must (a) be stable in structure and resistant to denaturation for a period of years or decades; (b) must be present without superaggregation in sufficient quantities to raise significantly the refractive index of the lens; and (c) must be small and discrete enough to enable lens transparency in the visible light spectrum. The recent discovery by Horwitz that a-crystallin is related in sequence to the heat shock protein family and that it can act in a chaperone-line fashion to prevent the superaggregation of partially denatured proteins may explain why it was """"""""recruited"""""""" as a lens protein. It is present in all the major non-lenticular tissues, but only in the lens are the two isoforms of a-crystallin found together. The long-term objective of this research is therefore to characterize the unique structural and functional properties of a-crystallin that contribute to long-term visual function and work against cataractogenesis. In the next grant period, the specific aims are (a) to investigate comparatively the structural and functional properties of native, reconstituted, and renatured a-crystallin aggregates in order to characterize the basis for their long-term stability; and (b) to compare the structural and functional properties of the a -crystallin isoforms in order to understand why it is only in the lens that both are found together. A variety of biophysical and physical biochemical techniques will be employed for this work, including circular dichroism spectropolarimetry to study secondary structure, fast performance liquid chromatography, fluorescence energy transfer, synchrotron scattering and diffraction, electron microscopy, and rheometry.
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