Project Summary The ⍺-crystallins (⍺A- and ⍺B- isoforms) are essential for the development and lifelong transparency of the eye lens. As molecular chaperones – members of the small heat shock protein family – the ⍺-crystallins play an integral part of the stability of lens proteostasis and preventing the formation of age-related opacities (i.e., protein aggregates) responsible for cataract formation. However, this system may become destabilized and/or overwhelmed as a result of the long-lived nature of lens proteins, and ultimately contribute to disease progression. Despite these fundamental roles in lens transparency and disease, there remains a critical gap in our understanding of the molecular mechanisms by which the ⍺-crystallins function, and how these proteins aggregate in response to aberrant (or age-related) conditions in the lens. A hallmark feature of the ⍺-crystallins is a remarkable degree of structural plasticity that is driven by sub-domain interactions involving flexible N- and C-termini, a feature that is thought to prevent crystallization under the highly concentrated environment of the eye lens. This molecular plasticity also contributes to ⍺-crystallin chaperone function, allowing it to adapt to and sequester a diverse range of destabilized proteins (aka clients) and prevent cytotoxic aggregation. However, these same features of intrinsic dynamics have stymied previous efforts to obtain the detailed structural characterizations (atomistic details) required to provide mechanistic insights into the function of these critical components of the eye lens. The aims of this proposal will leverage recent advances in the state-of-the-art methods of single particle CryoEM cryo-electron tomography (CryoET) – coupled with biophysical and functional studies – in order to obtain high-resolution structural information regarding the mechanism of ⍺-crystallin structural plasticity/polydispersity (Aim 1). Additional insights into the pathway(s) of chaperone/client co- aggregation (Aim 2) will be garnered from NSEM, light scattering, and crosslinking-MS/MS methods. Success of these aims will fill critical gaps in knowledge that have evaded the field for decades and provide detailed mechanistic insights into the molecular basis of ⍺-crystallin’s hallmark of structural plasticity and how the lens chaperone system becomes overwhelmed leading to cytotoxic protein aggregates such as those found in cataract.