Summary/abstract: Cataract is the leading cause of blindness worldwide, costing the US Healthcare system billions of dollars annually for surgical treatment. Lens opacification has been linked to dysfunction in major membrane proteins, Aquaporin-0 (AQP0) and Lim2. The overall goal of my project is to develop a comprehensive understanding of the structure and function of protein-protein and protein-lipid interactions of these membrane proteins. In the absence of a blood supply, the microcirculation system transports nutrients and removes wastes to inner fiber cells and is essential for maintaining lens transparency over decades of life; however, how this microcirculation system is established and maintained as a function of age is not well understood. Using advanced mass spectrometry techniques such as hydrogen-deuterium exchange-MS, native-MS and chemical crosslinking studies, I intend to elucidate how specific protein and lipid interactions impact the structure and function of AQP0 and Lim2; membrane proteins that are fundamental to the microcirculation system of the lens. The most abundant lens membrane protein, AQP0, plays important roles in lens fiber cell adhesion and water permeability with water permeability regulated by interaction with the calcium-binding protein, calmodulin. Data from my lab and others demonstrated that calmodulin interacts with the C-terminal tail of AQP0; however, molecular dynamic (MD) simulations suggest a non-canonical interaction with a cytosolic loop of AQP0. This MD prediction has not been experimentally validated. In addition to AQP0- protein interactions (Aim 1), I hypothesize that AQP0-lipid interactions (Aim 2) regulate AQP0 permeability and adhesion properties that underlie lens transparency; however, there are limited reports on AQP0 interactions with native lipids. Given the vital role of AQP0 in maintaining lens transparency and its connection to cataract, understanding regulatory interactions with proteins such as calmodulin and native lipids will clarify its role in the microcirculation system. The second most abundant membrane protein in the lens is Lim2 and, like AQP0, its mutation has been associated with cataractogenesis in mice; however, little is known about Lim2-protein interactions (Aim 1). Binding partners to Lim2 have been reported, i.e. calmodulin and galectin-3, but how these interactions modulate Lim2 structure and function are not clear. Additionally, native lens lipids have been reported to impact Lim2 subunit assembly, but the details underlying this phenomenon have not been explored. As a result of the scarcity of experimental research on Lim2-native lipid interactions (Aim 2), I will use native MS to identify specific lipid interactions and determine how they affect Lim2 structure. My findings will aid researchers develop therapeutic targets and/or practices that can prevent, reverse or delay cataract formation.