Unravelling Membrane Protein-Lipid Interactions using Nanodiscs and Mass Spectrometry

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PROJECT SUMMARY/ABSTRACT Membrane proteins are involved in many cellular processes and thus are critical drug targets for a wide range of diseases. However, there is a fundamental gap in understanding how the global changes to the lipid environment affect local membrane protein structure and function. Mounting evidence indicates that lipids can be essential for membrane protein function, but it is difficult to determine the molecular mechanisms underlying protein-lipid interactions. The primary challenge is that conventional structural biology tools and binding assays are poorly suited to characterizing transient and heterogeneous protein-lipid interactions. To advance our understanding of biological process and lay a foundation for advancing disease treatment, our goal is to develop new approaches to determine how lipid bilayers regulate membrane proteins. Studying a diverse set of membrane protein targets ranging from bacterial complexes to viral ion channels to human transporters, we are focused on answering several key questions. First, which lipids bind a given membrane protein target? Lipids are often observed in membrane protein structures, but it can be challenging to determine the identity of the lipids present in the local lipidome surrounding membrane proteins. We will use novel lipidomic lipid exchange-mass spectrometry methods to study enrichment of specific lipid species in nanodisc lipoprotein particles containing the membrane protein target. Our goal is to identify 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 membrane protein function, but it is often unclear where and how specifically they bind. We will develop new native mass spectrometry methods to determine the sites and selectivity of lipid binding to membrane protein targets. Our goal 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 are modulated in response to disease, age, and the environment, but it is unclear how these global lipid changes affect local membrane protein physiology. We will study the function of membrane protein targets in different lipid environments and with different mutants that affect lipid binding. For lipid sites that significantly affect function, 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 structure and function. This will impact biomedical research by identifying lipids important for maintaining protein activity and aiding in elucidating the physiological mechanisms of membrane proteins inside natural bilayers. Ultimately, an improved understanding of protein-lipid interactions holds the potential for improved drug discovery and for new therapeutic strategies for modulating membrane protein activity by modulating cellular lipids.