Project Summary Light-scattering opacities responsible for age-related cataracts are a result of aggregation and precipitation of the lens crystallins (α, β, and γ-crystallins). The α-crystallins (αA and αB isoforms) assemble as polydispersed oligomeric complexes and function as ATP-independent molecular chaperones (i.e., protein hold-ases). Both of these properties are thought to guard against aggregation events that would disrupt the delicate proteostasis of the lens. It is known that environmental stress and chemical modifications that accrue over our lifetimes destabilize the lens crystallins, and induce complex forms of protein-protein interactions that lead to aggregation (amorphous and potentially fibril). However, a major hurdle to understanding the aggregation pathways associated with cataracts, has been the lack of structural information on the major lens α-crystallins. This gap in knowledge is due to the lack of effective methods to characterize the inherently polydispersed structure of α-crystallin, the heterogeneity of chaperone-client aggregate formations, and evasiveness of fibril aggregation states identified under physiological conditions. In this proposal, we describe our multidisciplinary team-based approach, centered around the PI's expertise in the enabling technology of single particle CryoEM, that will finally allow us to interrogate the basis of α-crystallin molecular plasticity. Specifically, we aim to define high-resolution structures of the α-crystallins in their intrinsic polydispersed states (Aim 1), resolve key structural intermediates (aka “pre-aggregation states”) induced under saturating client conditions (Aim 2), and characterize a novel mechanism of fibrillogenesis discovered by our laboratory that is accessible to αB- crystallin under cellular conditions (Aim 3). Structural studies will be complimented by biophysical and functional characterization, performed in collaboration with Prof. Kirsten Lampi (OHSU), with the aim of illuminating mechanistic principles that define α-crystallin structure, polydispersity and stability – which are critical to avoidance of aggregation in the lens and therefore key to future success of drug-design strategies targeted at controlling age-related cataracts (and a range of other human crystallin-opathies).