PROJECT SUMMARY/ABSTRACT Membrane proteins are involved in many cellular processes and thus are critical drug targets for a wide rangeof diseases. However there is a fundamental gap in understanding how the global changes to the lipidenvironment affect local membrane protein structure and function. Mounting evidence indicates that lipids canbe essential for membrane protein function but it is difficult to determine the molecular mechanisms underlyingprotein-lipid interactions. The primary challenge is that conventional structural biology tools and binding assaysare poorly suited to characterizing transient and heterogeneous protein-lipid interactions. To advance our understanding of biological process and lay a foundation for advancing disease treatmentour goal is to develop new approaches to determine how lipid bilayers regulate membrane proteins. Studying adiverse set of membrane protein targets ranging from bacterial complexes to viral ion channels to humantransporters we are focused on answering several key questions. First which lipids bind a given membrane protein target? Lipids are often observed in membrane proteinstructures but it can be challenging to determine the identity of the lipids present in the local lipidome surroundingmembrane proteins. We will use novel lipidomic lipid exchange-mass spectrometry methods to study enrichmentof specific lipid species in nanodisc lipoprotein particles containing the membrane protein target. Our goal is toidentify unknown lipids that bind the membrane protein targets in complex mixtures of natural lipids. Second how and were do lipids interact with the protein? We know that lipids can be critical for membraneprotein function but it is often unclear where and how specifically they bind. We will develop new native massspectrometry methods to determine the sites and selectivity of lipid binding to membrane protein targets. Ourgoal is to uncover the molecular mechanisms driving lipid specificity at specific binding sites. Finally why are lipids important for membrane protein function? We know that bunk cellular lipids aremodulated in response to disease age and the environment but it is unclear how these global lipid changesaffect local membrane protein physiology. We will study the function of membrane protein targets in differentlipid environments and with different mutants that affect lipid binding. For lipid sites that significantly affectfunction we will perform structural analysis to connect lipid binding at specific sites with functional outcomes. Our overarching goal is to understand how global lipidomic changes affect local membrane protein structureand function. This will impact biomedical research by identifying lipids important for maintaining protein activityand aiding in elucidating the physiological mechanisms of membrane proteins inside natural bilayers. Ultimatelyan improved understanding of protein-lipid interactions holds the potential for improved drug discovery and fornew therapeutic strategies for modulating membrane protein activity by modulating cellular lipids.