A long-term objective of research in the PI's laboratory is to elucidate the molecular architecture of alpha-crystallin, the major protein component of lens-fiber cells, and to gain insight into the mechanism of its chaperone-like function. Protein-protein interactions between alphaA- and alphaB-crystallin are critical for the formation of the short-range spatial order responsible for lens transparency. By selectively binding unfolded proteins prone to aggregation, alphaA- and alphaB-crystallin delay the detrimental effect of age-dependent protein damage. This dual function is mediated by a dynamic oligomeric structure that endows these proteins with the ability to respond to changes in the cellular environment. The focus of this proposal is to test the hypothesis that the determinants of the quaternary-structure dynamic polymorphism are encoded in the N-terminal domain and to elucidate the structural and dynamic basis of molecular recognition and binding. A major goal (Aims 1 and 2) is to determine the secondary structure, tertiary folding pattern, and the quaternary interactions of the N-terminal domain in alphaA-crystallin. This, in conjunction with the structure of the C-terminal domain determined in the PI's laboratory, will provide the structural basis necessary for a mechanistic description of function. A second goal (Aim 3) is to dissect the chaperone function into well- characterized steps using thermodynamically destabilized substrate- proteins of known X-ray structure. In addition to obtaining the rate and stoichiometry of binding, experiments are proposed to identify the intermediate states recognized by alphaA-crystallin and to characterize the conformations that are stably bound. A third goal (Aim 4) is to construct protein chimeras of alphaA- crystallin, alphaB-crystallin and hsp 27, using homolog-scanning mutagenesis, in order to elucidate how sequence divergence in the N-terminal domain results in the structural, dynamic and functional differences between these proteins. These objectives will be achieved using extensive scanning mutagenesis in conjunction with novel functional assays, site-directed spin labeling and fluorescence spectroscopies. The results of the proposed research will shed light on how the lens responds to incipient protein damage that can lead to protein aggregation, the initial event in senile cataract. They will also elucidate, in a structural and dynamic context, the distinct roles of alphaA- and alphaB- crystallin, a critical step in the process of understanding the biophysics and biochemistry of lens transparency.
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