Cataracts affect the vision of 1 in 6 people over the age of 40 in the United States and most people by the age of 80. It is the leading cause of blindness worldwide. As a result, there is much interest in understanding the cause of cataracts and the mechanism by which they form. Cataracts are classified as a misfolding disease whereby degradation of the crystallin lens proteins leads to their aggregation and precipitation. However, critical structural and mechanistic information is lacking largely because it is experimentally difficult to obtain structural information about aggregated proteins and even more difficult to characterize intermediates that are responsible for precipitation. In this proposal, w will use a novel combination of 2D IR spectroscopy, isotope labeling, and mass spectrometry to uncover details about the structure and kinetic mechanism by which ?D- crystallin aggregates and the way in which the chaperone protein ?B-crystallin inhibits precipitation. Using expressed protein ligation to semi-synthesize ?D-crystallin, we will isotope label its individual domains so that their structures and kinetics can be monitored by 2D IR spectroscopy. Using UVB light to mimic covalent damage from solar radiation and initiate aggregation, we will monitor the kinetics and structures of the precipitates as they form. We know from our initial publications that acid-induced denaturation of ?D-crystallin leads solely to amyloid fiber formation with the C-terminal domain forming the fibril core, not the N-terminal domain as was previously thought. In contrast, preliminary results on UVB-denaturation reveal that covalent damage leads to both fibrillar and amorphous aggregates. Clearly, there is a competition between pathways that depends on the type of protein damage. Once these pathways are characterized, we will study how they are modified by the chaperone protein ?B-crystallin. For many proteins ?B-crystallin is a better chaperone against amorphous than fibrillar aggregates, but cataract deposits appear to be mostly amorphous aggregates, implying that the chaperone mechanism is quite different for the crystallin proteins. Finally, the in vitro mechanisms will be tested against in vivo protein extracs collected from human lenses. We want to know if naturally occurring damage or composition of the crystallins alters the in vitro mechanisms and/or structure of the precipitates. Preliminary results are shown for nearly every step in this proposal. Our novel approach of using semi-synthesis and 2D IR spectroscopy is providing molecular-level insights that are important to the large community of scientists devoted to understanding the chaperone mechanisms of ?B-crystallin and cataract formation.