Project Summary/Abstract Heterogeneity in spatial and temporal dimensions is a hallmark of mammalian cell membranes. This partitions cellular membranes into distinct nanodomains distinguished by specific lipid-lipid or lipid-protein interactions. These nanodomains provide a dynamic, spatiotemporally organized platform for membrane-localized regulation of critical signaling pathways, imparting unique organization and bioactivity to the signaling proteins (and their effectors/substrates) that are associated with or embedded in them. This regulation of membrane proteins by their endogenous microenvironment is fundamental to critical biological functions and is often impaired in diseases such as cancer, neurodegeneration, and immune disorders. Despite the significance of the native membrane milieu in modulating membrane protein functions, there is a scarcity of experimental approaches for studying membrane proteins in their native lipid environment with simultaneous spatial, temporal, and molecular resolution. Motivated by this inherent challenge, my goal is to develop an experimental platform to understand the hierarchical and functional organization of membrane proteins in an endogenous cell membrane environment with precise spatial and molecular resolution. I will use an amphipathic styrene-maleic acid copolymer (SMA and its chemical analogs) to excise circular patches of the cell membrane, generating ~10-15 nm-sized “native nanodiscs”. SMA-encapsulated membrane proteins of interest are surrounded by an annular ring of endogenous lipids and interacting proteins, preserving their local membrane microenvironments and offering unprecedented spatial resolution. Following further enrichment and purification, I will use single-molecule TIRF microscopy, native mass spectrometry, lipidomics and proteomics, functional assays, and structural studies to investigate the biophysical and biochemical properties of these SMA- encapsulated membrane proteins within their endogenous environment. I will study two clinically relevant membrane/membrane-associated proteins using this approach – (1) KRas, a small GTPase that is a prominent oncogene with >95% mutation frequency in pancreatic cancers, and (2) Trk family receptor tyrosine kinases that bind neurotrophins and are central to neuronal development, differentiation, and survival and are implicated in pain perception. I will address outstanding questions about the structural and functional organization of these proteins on the native membrane, how the spatially enriched proteome and lipidome around these proteins regulate their organization, how that influences the biological activity of these membrane proteins, and ultimately how that impacts downstream signaling. The direct involvement of protein- lipid interaction and dynamics in several diseases necessitates a quantitative understanding of membrane proteins in their native membrane milieu. My proposal outlines a general experimental pipeline with a broad and...