Role of Deamidation in Human Beta-Crystallin Structure

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DESCRIPTION (provided by applicant): Deamidation is the most prevalent post-translational modification in the lens, and increases with age. All of the major crystallins are deamidated, with the beta-crystallins the most heavily deamidated. Our laboratory has recently identified several specific in vivo sites of deamidation in crystallins allowing us to investigate the functional consequences of this modification. Several deamidation sites are in highly conserved regions between beta-subunits, suggesting that these deamidations occur in critical structural regions. However, the effects of deamidation on crystallin structure and stability are not known. The stability of crystallins is fundamental to the transparency of the lens. The formation of cataracts will only be understood when we begin to elucidate factors that alter crystallin stability. The long-term goal of our research is to determine the effects of deamidation on beta-crystallins in the human lens. The objective of this grant application is to characterize several specific in vivo sites of deamidation of beta-crystallins. We hypothesize that deamidation in critical regions disrupt structure and stability promoting insolubilization, while deamidations in non-critical regions foster solubility. Our hypothesis is derived from our previous studies showing that a deamidation at the interface between domains decreased stability, while deamidations at other sites increased stability, or had little effect. To test our hypothesis, we will use site directed mutagenesis to introduce in vivo deamidations in regions predicted to be structurally significantly. We will then characterize their structure, stability, and tendency to aggregate using advanced biophysical techniques, such as multi-angle laser light scattering, and dynamic light scattering. Only after we understand how deamidation alters crystallin structure can we correlate specific deamidations to cataract formation. We can then begin to investigate methods to prevent deamidation or design chaperones that prevent structural changes at these sites.