Understanding how membrane composition directs membrane protein structure and function

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The goal of this interdisciplinary team science proposal is to extend and inform biochemical and structural biology approaches for studying membrane proteins by understanding how their native environments define structure and function. To date, the majority of mechanistic studies of integral membrane proteins (IMPs) have not captured the properties and functional contributions of the membranes in which the IMPs are embedded. The central goal of our collaborative team is to develop new technologies and approaches that will allow us to: i.) Define lipid components and protein co-receptor components of functional complexes; ii.) Evaluate the role of the local membrane environment in function and regulation of the IMPs; and iii.) Determine the structures of these assemblies. Driven – and made possible – by the recruitment of six key junior faculty to the Departments of Pharmacology and Cell Biology over the past 5 years, we have assembled an interdisciplinary team with shared interest in transmembrane protein structure and function. Our team members bring complementary expertise to the project with skills in cryo electron microscopy (cryoEM), top-down and bottom-up mass spectrometry (MS), multi-omic analysis, optical imaging, biochemistry, and cellular signaling. We are very well placed to make unique advances in understanding membrane proteins involved in regulation of bacterial lipopolysaccharide synthesis, insect olfaction, mammalian ion channels, and mammalian receptors in the G protein-couple receptor (GPCR), Frizzled, and receptor tyrosine kinase families. Our Specific Aims are: 1: Identify native environments of integral membrane proteins To achieve this, we will identify new membrane-active copolymers that efficiently extract IMPs of interest, and use state-of-the-art lipidomics, proteomics, and native mass spectrometry to elucidate the molecular components of the protein’s membrane environment. 2: Understand how the native membrane environment modulates or determines membrane protein function Using a wide variety of assays – tailored to each IMP – we will ask how the specific membrane environment identified in Aim 1 influences IMP activity and oligomerization. We will also use limited proteolysis and H/D exchange mass spectrometry approaches to assess the influence of the membrane composition on conformation and structural dynamics 3: Determine structures of membrane proteins and complexes in native membrane environments We will determine structures of the target IMPs in defined membrane environments using cryoEM, to ask how known specific IMP-associated lipids and other components interact with and modulate IMP structure.