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Grant

Unravelling Membrane Protein-Lipid Interactions using Nanodiscs and Mass Spectrometry

Sponsored by National Institute of General Medical Sciences

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$1.9M Funding
1 People
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Abstract

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 lipid environment affects membraneprotein structure and function. Mounting evidence indicates that lipids can be essential for membrane proteinfunction but it is challenging to determine the molecular mechanisms underlying the importance of protein-lipidinteractions. The primary challenge is that conventional structural biology tools and binding assays are poorlysuited to characterizing transient and heterogeneous protein-lipid interactions. To understand how lipids modulate membrane protein structure and function my research program isdeveloping new tools to study protein-lipid interactions by combining lipoprotein nanodiscs with massspectrometry (MS). These new technologies will be developed using well-characterized bacterial membraneprotein complexes before being applied to more complex mammalian proteins such as rhodopsin and uncouplingprotein 2. Our goal is to answer four questions for a given membrane protein target. 1) What lipids interact withthe target? To identify the endogenous lipids that surround the target we are developing a hybridlipopeptide/lipoprotein approach to solubilize membranes surrounded by their natural lipids into nanodiscswithout the need for detergent. Following purification of the target we will extract and identify the lipids that arenaturally associated with the target. 2) How strongly do lipids bind to the target? To distinguish tightly boundlipids from weakly associated lipids we will assemble lipoprotein nanodiscs with different mixtures of lipids anduse native MS to ionize the intact nanodisc assembly. Using collisional activation we will gradually dissociatethe nanodisc to measure the composition of the lipid annular belt and tightly associated structural lipids.Furthermore we will use lipid exchange between nanodiscs to measure lipid binding constants in an experimentanalogous to equilibrium dialysis. 3) Where do lipids interact with the protein structure? After predicting lipid-binding sites using molecular dynamics (MD) we will test the predictions by mutating interacting residues andusing native MS to detect the disruption of the binding sites. 4) Why are specific protein-lipid interactionsimportant for function? By pairing MD mutagenesis and native MS with functional studies we will connectlipid-dependent effects on protein function with specific lipid binding sites. Our overarching goal is to develop a toolbox for unravelling the molecular mechanisms of protein-lipidinteractions. 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 withmembrane protein targets and for new therapeutic strategies for modulating membrane protein activity.

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